N-(aryl/heteroaryl) amino acid esters, pharmaceutical compositions comprising same, and methods for inhibiting alpha- amyloid peptide release and/or its synthesis by use of such compounds

ABSTRACT

Disclosed are compounds which inhibit β-amyloid peptide release and/or its synthesis, and, accordingly, have utility in treating Alzheimer&#39;s disease. Also disclosed are pharmaceutical compositions comprising a compound which inhibits β-amyloid peptide release and/or its synthesis as well as methods for treating Alzheimer&#39;s disease both prophylactically and therapeutically with such pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application No. 08/975,977, filedNov. 21, 1997, now U.S. Pat. No. 5,965,614, which claims the benefit ofprovisional Application No. 60/104,593, filed Nov. 22, 1996, which wasconverted pursuant to 37 C.F.R. §1.53(b)(2)(ii) from U.S. patentapplication No. 08/755,444, filed Nov. 22, 1996.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

This invention relates to compounds which inhibit β-amnyloid peptiderelease and/or its synthesis, and, accordingly, have utility in treatingAlzheimer's disease.

REFERENCES

The following publications, patents and patent applications are cited inthis application as superscript numbers:

1 Glenner, et al., “Alzheimier's Disease: Initial Report of thePurification and Characterization of a Novel Cerebrovascular AmyloidProtein”, Biochem. Biophys. Res. Commun., 120:885-890 (1984).

2 Glenner, et al., “Polypeptide Marker for Alzheimner's Disease and itsUse for Diagnosis”, U.S. Pat. No. 4,666,829 issued May 19, 1987.

3 Selkoe, “The Molecular Pathology of Alzheimer's Disease”, Neuron,6:487-498 (1991).

4 Goate, et al., “Segregation of a Missense Mutation in the AmyloidPrecursor Protein Gene with Familial Alzheimer's Disease”, Nature,349:704-706 (1990).

5 Chartier-Harlan, et al., “Early-Onset Alzheimer's Disease Caused byMutations at Codon 717 of the β-Amyloid Precursor Proteing Gene”,Nature, 353:844-846 (1989).

6 Murrell, et al., “A Mutation in the Amyloid Precursor ProteinAssociated with Hereditary Alzheimer's Disease”, Science, 254:97-99(1991).

7 Mullan, et al., “A Pathogenic Mutation for Probable Alzheimer'sDisease in the APP Gene at the N-Terminus of β-Amyloid, Nature Genet.,1:345-347 (1992).

8 Schenk, et al., “Methods and Compositions for the Detection of Solubleβ-Amyloid Peptide”, International Patent Application Publication No. WO94/10569, published May 11, 1994.

9 Selkoe, “Amyloid Protein and Alzheimer's Disease”, ScientificAmerican, pp. 2-8, November, 1991.

10 Yates, et al., “N,N-Disubstituted Amino Acid Herbicides”, U.S. Pat.No. 3,598,859, issued Aug. 10, 1971.

11 Losse, et al., Tetrahedron, 27:1423-1434 (1971).

12 Citron, et al., “Mutation of the β-Amyloid Precursor Protein inFamilial Alzheimer's Disease Increases β-Protein Production, Nature,360:672-674 (1992).

13 Hansen, et al., “Reexamination and Further Development of a Preciseand Rapid Dye Method for Measuring Cell Growth/Cell Kill”, J. Immun.Meth., 119:203-210 (1989).

All of the above publications, patents and patent applications areherein incorporated by reference in their entirety to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

STATE OF THE ART

Alzheimer's Disease (AD) is a degenerative brain disorder characterizedclinically by progressive loss of memory, cognition, reasoning, judgmentand emotional stability that gradually leads to profound mentaldeterioration and ultimately death. AD is a very common cause ofprogressive mental failure (dementia) in aged humans and is believed torepresent the fourth most common medical cause of death in the UnitedStates. AD has been observed in races and ethnic groups worldwide andpresents a major present and future public health problem. The diseaseis currently estimated to affect about two to three million individualsin the United States alone. AD is at present incurable. No treatmentthat effectively prevents AD or reverses its symptoms and course iscurrently known.

The brains of individuals with AD exhibit characteristic lesions termedsenile (or amyloid) plaques, amyloid angiopathy (amyloid deposits inblood vessels) and neurofibrillary tangles. Large numbers of theselesions, particularly amyloid plaques and neurofibrillary tangles, aregenerally found in several areas of the human brain important for memoryand cognitive function in patients with AD. Smaller numbers of theselesions in a more restrictive anatomical distribution are also found inthe brains of most aged humans who do not have clinical AD. Amyloidplaques and amyloid angiopathy also characterize the brains ofindividuals with Trisomy 21 (Down's Syndrome) and Hereditary CerebralHemorrhage with Amyloidosis of the Dutch Type (HCHWA-D). At present, adefinitive diagnosis of AD usually requires observing the aforementionedlesions in the brain tissue of patients who have died with the diseaseor, rarely, in small biopsied samples of brain tissue taken during aninvasive neurosurgical procedure.

The principal chemical constituent of the amyloid plaques and vascularamyloid deposits (amyloid angiopathy) characteristic of AD and the otherdisorders mentioned above is an approximately 4.2 kilodalton (kD)protein of about 39-43 amino acids designated the β-amyloid peptide(βAP) or sometimes Aβ, AβP or β/A4. β-Amyloid peptide was first purifiedand a partial amino acid sequence was provided by Glenner, et al.¹ Theisolation procedure and the sequence data for the first 28 amino acidsare described in U.S. Pat. No. 4,666,829².

Molecular biological and protein chemical analyses have shown that theβ-amyloid peptide is a small fragment of a much larger precursor protein(APP), that is normally produced by cells in many tissues of variousanimals, including humans. Knowledge of the structure of the geneencoding the APP has demonstrated that β-amyloid peptide arises as apeptide fragment that is cleaved from APP by protease enzyme(s). Theprecise biochemical mechanism by which the β-amyloid peptide fragment iscleaved from APP and subsequently deposited as amyloid plaques in thecerebral tissue and in the walls of the cerebral and meningeal bloodvessels is currently unknown.

Several lines of evidence indicate that progressive cerebral depositionof β-amyloid peptide plays a seminal role in the pathogenesis of AD andcan precede cognitive symptoms by years or decades. See, for example,Selkoe³. The most important line of evidence is the discovery thatmissense DNA mutations at amino acid 717 of the 770-amino acid isoformof APP can be found in affected members but not unaffected members ofseveral families with a genetically determined (familial) form of AD(Goate, et al.⁴; Chartier Harlan, et al.⁵; and Murrell, et al.⁶) and isreferred to as the Swedish variant. A double mutation changinglysine⁵⁹⁵-methionine⁵⁹⁶ to asparagine⁵⁹⁵-leucine⁵⁹⁶ (with reference tothe 695 isoform) found in a Swedish family was reported in 1992 (Mullan,et al.⁷). Genetic linkage analyses have demonstrated that thesemutations, as well as certain other mutations in the APP gene, are thespecific molecular cause of AD in the affected members of such families.In addition, a mutation at amino acid 693 of the 770-amino acid isoformof APP has been identified as the cause of the β-amyloid peptidedeposition disease, HCHWA-D, and a change from alanine to glycine atamino acid 692 appears to cause a phenotype that resembles AD is somepatients but HCHWA-D in others. The discovery of these and othermutations in APP in genetically based cases of AD prove that alterationof APP and subsequent deposition of its β-amyloid peptide fragment cancause AD.

Despite the progress which has been made in understanding the underlyingmechanisms of AD and other β-amyloid peptide related diseases, thereremains a need to develop methods and compositions for treatment of thedisease(s). Ideally, the treatment methods would advantageously be basedon drugs which are capable of inhibiting β-amyloid peptide releaseand/or its synthesis in vivo.

SUMMARY OF THE INVENTION

This invention is directed to the discovery of a class of compoundswhich inhibit β-amyloid peptide release and/or its synthesis and,therefore, are useful in the prevention of AD in patients susceptible toAD and/or in the treatment of patients with AD in order to inhibitfurther deterioration in their condition. The class of compounds havingthe described properties are defined by formula I below:

wherein

R¹ is selected from the group consisting of:

(a) a substituted phenyl group of formula II:

 wherein

R^(c) is selected from the group consisting of acyl, alky, alkoxy,alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen, nitro,trihalomethyl, thioalkoxy, and wherein R^(b) and R^(c) are fused to forma heteroaryl or heterocyclic ring with the phenyl ring wherein theheteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from1 to 3 are heteroatoms independently selected from the group consistingof oxygen, nitrogen and sulfur;

R^(b) and R^(b′) are independently selected from the group consisting ofhydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy withthe proviso that R^(b), R^(b′) and R^(c) are not all hydrogen and withthe further proviso that when R^(c) is hydrogen, then neither R^(b) norR^(b′) are hydrogen;

(b) 2-naphthyl; and

(c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1to 5 substituents selected from the group consisting of alkyl, alkoxy,halo, cyano, nitro, trihalomethyl, and thioalkoxy;

R² is selected from the group consisting of hydrogen, alkyl of from 1 to4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms andalkylthioalkoxy of from 1 to 4 carbon atoms; and

R³ is selected from the group consisting of:

(a) —Y(CH₂)_(n)CHR⁴R⁵ wherein n is an integer of from 0 to 2, Y isselected from the group consisting of oxygen and sulfur, R⁴ and R⁵ areindependently selected from the group consisting of hydrogen, alkyl,alkenyl, aryl optionally substituted with from 1 to 3 substituentsselected from the group consisting of alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy, heteroaryl optionally substituted withfrom 1 to 3 substituents selected from the group consisting of alkyl,alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy, and where R⁴and R⁵ are joined to form a cycloalkyl group, a cycloalkenyl group, or aheterocyclic group;

(b) —ON═C(NH₂)R⁶ where R⁶ is selected from the group consisting ofalkyl, aryl, cycloalkyl, and heteroaryl;

(c) —O(CH₂)_(p)C(O)OR⁷ wherein p is an integer of from 1 to 2 and R⁷ isalkyl;

(d) —NR⁸R⁹ wherein R⁸ and R⁹ are joined to form a pyrrolyl group; and

pharmaceutically acceptable salts thereof

with the provisos that

1. when R¹ is the substituted phenyl group of formula II above, R^(b′)is hydrogen, R^(b) and R^(c) are chloro, and R² is methyl, then R³ isnot —OCH(CH₃)-φ;

2. when R¹ is the substituted phenyl group of formula II above, whenR^(b′) is hydrogen, R^(b) and R^(c) are chloro, and R³ is —OCH₂CH₃ thenR² is not hydrogen;

3. when R¹ is the substituted phenyl group of formula II above, R^(b′)is hydrogen, R^(b) and R^(c) are chloro, and R³ is —OCH₂CH(CH₃)₂ then R²is not —CH(CH₃)CH₂CH₃; and

4. when R¹ is N-methylindol-5-yl and R² is methyl, then R³ is not—OCH₂CH₃.

Surprisingly, any substituents at the 2 and/or 6 positions orsubstituents at the 3, 4 and/or 5 positions, other than thosespecifically specified above, eliminate the ability of the resultingcompounds to inhibit β-amyloid peptide release and/or its synthesis.

Accordingly, in one of its method aspects, this invention is directed toa method for inhibiting β-amyloid peptide release and/or its synthesisin a cell which method comprises administering to such a cell an amountof a compound or a mixture of compounds of formula I above effective ininhibiting the cellular release and/or synthesis of β-amyloid peptide.

Because the in vivo generation of β-amyloid peptide is associated withthe pathogenesis of AD^(8,9), the compounds of formula I can also beemployed in conjunction with a pharmaceutical composition toprophylactically and/or therapeutically prevent and/or treat AD.Accordingly, in another of its method aspects, this invention isdirected to a prophylactic method for preventing the onset of AD in apatient at risk for developing AD which method comprises administeringto said patient a pharmaceutical composition comprising apharmaceutically inert carrier and an effective amount of a compound ora mixture of compounds of formula I above.

In yet another of its method aspects, this invention is directed to atherapeutic method for treating a patient with AD in order to inhibitfurther deterioration in the condition of that patient which methodcomprises administering to said patient a pharmaceutical compositioncomprising a pharmaceutically inert carrier and an effective amount of acompound or a mixture of compounds of formula I above.

In formula I above, R¹ substituted phenyls are preferably 4-substituted,3,5-disubstituted or 3,4-disubstituted phenyl substituents wherein thesubstituents at the 3 and/or 5 positions are defined by R^(b), R^(b′) asabove and the substituent at the 4 position is defined by R^(c) asabove. Particularly preferred 3,5-disubstituted phenyls include, by wayof example, 3,5-dichlorophenyl, 3,5-difluorophenyl,3,5-di(trifluoromethyl)phenyl, 3,5-dimethoxyphenyl, and the like.Particularly, preferred 3,4-disubstituted phenyls include, by way ofexample, 3,4-dichlorophenyl, 3,4-difluorophenyl,3-(trifluoromethyl)-4-chlorophenyl, 3-chloro-4-cyanophenyl,3-chloro-4-iodophenyl, 3,4-methylenedioxyphenyl,3,4-ethylenedioxyphenyl, and the like. Particularly preferred4-substituted phenyls include, by way of example, 4-azidophenyl,4-bromophenyl, 4-chlorophenyl, 4-cyanophenyl, 4-ethylphenyl,4-fluorophenyl, 4-iodophenyl, 4-(phenylcarbonyl)phenyl,4-(1-ethoxy)ethylphenyl, 4-(ethoxycarbonyl)phenyl, and the like.

Other preferred R¹ substituents include, by way of example, 2-naphthyl,2-methylquinolin-6-yl, benzothiazol-6-yl, 5-indolyl, and the like.

Preferably R² is selected from the group consisting of alkyl of from 1to 4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms andalkylthioalkoxy of from 1 to 4 carbon atoms. Particularly preferred R²substituents include, by way of example, methyl, ethyl, n-propyl,iso-butyl, and the like.

Preferred R³ substituents include methoxy, ethoxy, iso-propoxy,n-propoxy, n-butoxy, iso-butoxy, cyclopentoxy, allyloxy,4-methylpentoxy, —O—CH₂-(2,2-dimethyl-1,3-dioxolan-4-yl),—O—CH₂-cyclohexyl, —O—CH₂-(3-tetrahydrofuranyl),—O—CH₂—C(O)O-tert-butyl, —O—CH₂—C(CH₃)₃, —O—CH₂-φ, —OCH₂CH(CH₂CH₃)₂,—O(CH₂)₃CH(CH₃)₂, —ON═C(NH₂)φ, —ON═C(NH₂)CH₃, —ON═C(NH₂)CH₂CH₃,—ON═C(NH₂)CH₂CH₂CH₃, —ON═C(NH₂)-cyclopropyl, —ON═C(NH₂)—CH₂-cyclopropyl,—ON═C(NH₂)-cyclopentyl, —ON═C(NH₂)CH₂CH(CH₃)₂, and the like.

This invention also provides for novel pharmaceutical compositionscomprising a pharmaceutically inert carrier and a compound of theformula I above.

Particularly preferred compounds for use in the methods and compositionsof this invention include, by way of example, the following wherein thestereochemistry of the R² group (where appropriate) is derived from theL-amino acid:

N-(3,4-dichlorophenyl)alanine ethyl ester;

N-(3-trifluoromethyl-4-chlorophenyl)alanine ethyl ester;

N-(3,5-dichlorophenyl)alanine ethyl ester;

N-(3,4-difluorophenyl)alanine ethyl ester;

N-(3,4-dichlorophenyl)alanine benzyl ester;

N-(3,4-dichlorophenyl)alanine iso-butyl ester;

N-(3,4-dichlorophenyl)alanine iso-propyl ester;

N-(3,4-dichlorophenyl)alanine n-butyl ester;

N-(3,4-dichlorophenyl)alanine methyl ester;

N-(3,4-dichlorophenyl)alanine cyclopentyl ester;

N-(3,4-dichlorophenyl)alanine n-propyl ester;

N-(3,4-dichlorophenyl)alanine allyl ester;

N-(3,4-dichlorophenyl)alanine 4-methylpentyl ester;

N-(3,4-dichlorophenyl)alanine 2,2-dimethyl-1,3-dioxolane-4-methyl ester;

N-(3,4-dichlorophenyl)alanine cyclohexylmethyl ester;

N-(3,4-dichlorophenyl)alanine tert-butoxycarbonylmethyl ester;

N-(3,4-dichlorophenyl)leucine iso-butyl ester;

2-[N-(3,4-dichlorophenyl)amino]pentanoic acid iso-butyl ester;

N-(4-cyanophenyl)alanine iso-butyl ester;

N-(3-chloro-4-cyanophenyl)alanine iso-butyl ester;

N-(3,4-dichlorophenyl)alanine tetrahydrofuran-3-yl-methyl ester;

N-(3-chloro-4-iodophenyl)alanine iso-butyl ester;

2-[N-(3,4-dichlorophenyl)amino]butanoic acid isobutyl ester;

N-(4-chlorophenyl)alanine iso-butyl ester;

N-(3,5-dichlorophenyl)alanine iso-butyl ester;

N-(4-ethylphenyl)alanine methyl ester;

N-[4-(1-ethoxy)ethylphenyl]alanine methyl ester;

N-(3,4-dichlorophenyl)alanine 2,2-dimethylpropyl ester;

N-(3,4-dichlorophenyl)glycine iso-butyl ester;

N-(3,4-dichlorophenyl)alanine 2-ethylbutyl ester;

N-(3-chloro-4-iodophenyl)alanine iso-butyl ester;

N-(4-azidophenyl)alanine iso-butyl ester;

N-[(4-phenylcarbonyl)phenyl]alanine iso-butyl ester;

N-(3,5-difluorophenyl)alanine iso-butyl ester;

N-(3,4-dichlorophenyl)alanine O-acylacetamidoxime ester,

N-(3,4-dichlorophenyl)alanine pyrrolyl amide;

N-(3,4-dichlorophenyl)alanine O-acylpropionamideoxime ester;

N-(3,4-dichlorophenyl)alanine O-acylbutyramideoxime ester;

2-[N-(naphth-2-yl)amino]butanoic acid ethyl ester;

N-(naphth-2-yl)alanine iso-butyl ester;

N-(2-methylquinolin-6-yl)alanine iso-butyl ester;

N-(3,4-ethylenedioxyphenyl)alanine iso-butyl ester;

N-(3,4-methylenedioxyphenyl)alanine iso-butyl ester;

N-(naphth-2-yl)alanine methyl ester;

N-(benzothiazol-6-yl)alanine ethyl ester;

N-(indol-5-yl)alanine iso-butyl ester;

N-(naphth-2-yl)alanine O-acylacetamidoxime ester;

N-(2-naphthyl)alanine ethyl ester;

N-(4-ethoxycarbonylphenyl)alanine iso-butyl ester;

N-(3,5-di(trifluoromethyl)phenyl)alanine iso-butyl ester;

N-(3,5-dimethoxyphenyl)alanine iso-butyl ester;

N-(2-napthyl)alanine O-acylpropionamidoxime ester;

N-(2-napthyl)alanine O-acylbutyramidoxime ester;

N-(2-napthyl)alanine O-acylisovaleramidoxie ester;

N-(2-napthyl)alanine O-acylbenzamidoxime ester;

N-(2-napthyl)alanine O-acylcyclopropanecarboxamidoxime ester;

N-(2-napthyl)alanine O-acylcyclopropylacetamidoxime ester; and

N-(2-napthyl)alanine O-acylcyclopentanecarboxamidoxime ester.

Still further, this invention provides for novel compounds of theformula III:

wherein

R¹ is selected from the group consisting of:

(a) a substituted phenyl group of formula II:

 wherein

R^(c) is selected from the group consisting of acyl, alkyl, alkoxy,alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen, nitro,trihalomethyl, thioalkoxy, and where R^(b) and R^(c) are fused to form aheteroaryl or heterocyclic ring with the phenyl ring wherein theheteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from1 to 3 are heteroatoms independently selected from the group consistingof oxygen, nitrogen and sulfur;

R^(b) and R^(b′) are independently selected from the group consisting ofhydrogen, halo, nitro, cyano, trihalomethyl, alkoxy, and thioalkoxy withthe proviso that R^(b), R^(b′) and R^(c) are not all hydrogen and withthe further proviso that when R^(c) is hydrogen, then neither R^(b) norR^(b′) are hydrogen;

(b) 2-naphthyl; and

(c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1to 5 substituents selected from the group consisting of alkyl, alkoxy,halo, cyano, nitro, trihalomethyl, and thioalkoxy;

R² is selected from the group consisting of hydrogen, alkyl of from 1 to4 carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms andalkylthioalkoxy of from 1 to 4 carbon atoms; and

R³ is selected from the group consisting of:

(a) —Y(CH₂)_(n)CHR⁴R⁵ wherein n is an integer of from 0 to 2, Y isselected from the group consisting of oxygen and sulfur, R⁴ and R⁵ areindependently selected from the group consisting of hydrogen, alkyl,alkenyl, aryl optionally substituted with from 1 to 3 substituentsselected from the group consisting of alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy, heteroaryl optionally substituted withfrom 1 to 3 substituents selected from the group consisting of alkyl,alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy, and where R⁴and R⁵ are joined to form a cycloalkyl group, a cycloalkenyl group or aheterocyclic group;

(b) —ON═C(NH₂)R⁶ where R⁶ is selected from the group consisting ofalkyl, aryl, cycloalkyl, and heteroaryl;

(c) —O(CH₂)_(p)C(O)OR⁷ wherein p is an integer of from 1 to 2 and R⁸ isalkyl; and

(d) —NR⁸R⁹ wherein R⁸ and R⁹ are joined to form a pyrrolyl group;

and pharmaceutically acceptable salts thereof

with the proviso excluding the following compounds:

1. when R¹ is the substituted phenyl group of formula II above, R^(b′)is hydrogen, R^(b) and R^(c) are chloro, and R² is methyl, then R³ isnot —OCH(CH₃)-φ;

2. when R¹ is the substituted phenyl group of formula II above, whenR^(b′) is hydrogen, R^(b) and R^(c) are chloro, and R³ is —OCH₂CH₃ thenR² is not hydrogen;

3. when R¹ is the substituted phenyl group of formula II above, R^(b′)is hydrogen, R^(b) and R^(c) are chloro, and R³ is —OCH₂CH(CH₃)₂ then R²is not —CH(CH₃)CH₂CH₃; and

4. when R¹ is N-methylindol-5-yl and R² is methyl, then R³ is not—OCH₂CH₃;

and still with further proviso excluding the following known compounds:N-(4-chlorophenyl)alanine ethyl ester; N-(3,4-dichlorophenyl)alanineethyl ester; N-(3,5-dichlorophenyl)alanine ethyl ester;N-(4-n-butylphenyl)alanine ethyl ester; N-(3,4-dinitrophenyl)alanineethyl ester; N-(4-chlorophenyl)glycine heptenyl ester;N-(4-methylphenyl)glycine butyl ester; N-(3-nitrophenyl)glycine decylester; N-(3,4-difluorophenyl)alanine methyl ester;N-(3,4difluorophenyl)alanine ethyl ester; N-(3,4-difluorophenyl)alanineiso-propyl ester; N-(4-fluorophenyl)alanine ethyl ester;N-(3-chloro-4-fluorophenyl)alanine methyl ester;N-(3-chloro-4-fluorophenyl)alanine ethyl ester; andN-(3-chloro-4-fluorophenyl)alanine iso-propyl ester.

Preferred compounds of formula I above include those set forth inFormula IV below:

R^(b) R^(c) R^(b′) R² R³ —CF₃ —Cl H —CH₃ —OCH₂CH₃ —Cl —Cl H —CH₃—OCH₂CH₃ —Cl —Cl H —CH₃ —OCH₂-φ —Cl —Cl H —CH₃ —OCH₂CH(CH₃)₂ —Cl —Cl H—CH₃ —OCH(CH₃)₂ —Cl —Cl H —CH₃ —O(CH₂)₃CH₃ —Cl —Cl H —CH₃ —OCH₃ —Cl —ClH —CH₃ —O-cyclopentyl —Cl —Cl H —CH₃ —OCH₂CH₂CH₃ —Cl —Cl H —CH₃ —O-allyl—Cl —Cl H —CH₃ —O(CH₂)₃CH(CH₃)₂ —Cl —Cl H —CH₃—O—CH₂-(2,2-dimethyl-1,3-dioxolan-4-yl) —Cl —Cl H —CH₃ —OCH₂-cyclohexyl—Cl —Cl H —CH₃ —OCH₂C(O)O-tert-butyl —Cl —Cl H —CH₂CH(CH₃)₂—OCH₂CH(CH₃)₂ —Cl —Cl H —CH₂CH₂CH₃ —OCH₂CH(CH₃)₂ H —CN H —CH₃—OCH₂CH(CH₃)₂ —Cl —CN H —CH₃ —OCH₂CH(CH₃)₂ —Cl —Cl H —CH₃ —OCH₂CH(CH₃)₂—Cl —Cl H —CH₃ —OCH₂-(3-tetra-hydrofuranyl) —Cl —I H —CH₃ —OCH₂CH(CH₃)₂—Cl —Cl H —CH₂CH₃ —OCH₂CH(CH₃)₂ H —Cl H —CH₃ —OCH₂CH(CH₃)₂ —Cl H —Cl—CH₃ —OCH₂CH(CH₃)₂ H —CH₂CH₃ H —CH₃ —OCH₃ H —CH(CH₃)—OC₂H₅ H —CH₃ —OCH₃—Cl —Cl H —CH₃ —OCH₂C(CH₃)₄ —Cl —Cl H —H —OCH₂CH(CH₃)₂ —Cl —Cl H —CH₃—OCH₂CH(CH₂CH₃)₂ —Cl —I H —CH₃ —OCH₂CH(CH₃)₂ H azido H —CH₃—OCH₂CH(CH₃)₂ H —C(O)φ H —CH₃ —OCH₂CH(CH₃)₂ —F H —F —CH₃ —OCH₂CH(CH₃)₂—Cl —Cl H —CH₃ —O—N═C(NH₂)CH₃ —Cl —Cl H —CH₃ N-pyrrolyl H —CO₂CH₂CH₃ H—CH₃ —OCH₂CH(CH₃)₂ —CF₃ H —CF₃ —CH₃ —OCH₂CH(CH₃)₂ —Cl —Cl H —CH₃—ON═C(NH₂)—CH₂CH₃ —Cl —Cl H —CH₃ —ON═C(NH₂)—CH₂CH₂CH₃ —OCH₃ H —OCH₃ —CH₃—OCH₂CH(CH₃)₂

Other preferred compounds of formula I include those set forth in thefollowing formula V:

R¹ R² R³ 2-naphthyl —CH₂CH₃ —OCH₂CH₃ 2-naphthyl —CH₃ —OCH₂CH(CH₃)₂2-methylquinolin- —CH₃ —OCH₂CH(CH₃)₂ 3-yl 3,4-ethylene- —CH₃—OCH₂CH(CH₃)₂ dioxyphenyl 3,4-methylene- —CH₃ —OCH₂CH(CH₃)₂ dioxyphenyl2-naphthyl —CH₃ —OCH₃ benzothiazol-6-yl —CH₃ —OCH₂CH₃ 5-indolyl —CH₃—OCH₂CH(CH₃)₂ 2-naphthyl —CH₃ —O—N═C(NH₂)CH₃ 2-naphthyl —CH₃ —OCH₂CH₃2-naphthyl —CH₃ —O—N═C(NH₂)CH₂CH₃ 2-naphthyl —CH₃ —O—N═C(NH₂)CH₂CH₂CH₃2-naphthyl —CH₃ —O—N═C(NH₂)CH₂CH(CH₃)₂ 2-naphthyl —CH₃ —O—N═C(NH₂)φ2-naphthyl —CH₃ —O—N═C(NH₂)-cyclopropyl 2-naphthyl —CH₃—O—N═C(NH₂)CH₂-cyclopropyl 2-naphthyl —CH₃ —O—N═C(NH₂)-cyclopentyl

DETAILED DESCRIPTION OF THE INVENTION

As above, this invention relates to compounds which inhibit β-amyloidpeptide release and/or its synthesis, and, accordingly, have utility intreating Alzheimer's disease. However, prior to describing thisinvention in further detail, the following terms will first be defined.

Definitions

The term “β-amyloid peptide” refers to a 39-43 amino acid peptide havinga molecular weight of about 4.2 kD, which peptide is substantiallyhomologous to the form of the protein described by Glenner, et al.¹including mutations and post-translational modifications of the normalβ-amyloid peptide. In whatever form, the β-amyloid peptide isapproximately a 39-43 amino acid fragment of a large membrane-spanningglycoprotein, referred to as the β-amyloid precursor protein (APP). Its43-amino acid sequence is:

1

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr

11

Glu Val His His Gln Lys Leu Val Phe Phe

21

Ala Glu Asp Val Gly Ser Asn Lys Gly Ala

31

Ile Ile Gly Leu Met Val Gly Gly Val Val

41

Ile Ala Thr (SEQ ID NO: 1)

or a sequence which is substantially homologous thereto.

“Alkyl” refers to monovalent alkyl groups preferably having from 1 to 10carbon atoms and more preferably 1 to 6 carbon atoms. This term isexemplified by groups such as methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, n-hexyl, and the like.

“Alkylene” refers to divalent alkylene groups preferably having from 1to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This term isexemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkaryl” refers to -alkylene-aryl groups preferably having from 1 to 10carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in thearyl moiety. Such alkaryl groups are exemplified by benzyl, phenethyland the like.

“Alkoxy” refers to the group “alkyl-O—” where alkyl is as definedherein. Preferred alkoxy groups include, by way of example, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

“Alkoxycarbonyl” refers to the group “alkyl-O—C(O)—” wherein alkyl is asdefined herein. Such groups include, by way of example, methoxycarbonyl,ethoxycarbonyl, n-propoxycarbonyl, iso-propoxycarbonyl,n-butoxycarbonyl, tert-butoxyfarbonyl, sec-butoxycarbonyl,n-pentoxycarbonyl, n-hexoxycarbonyl, and the like.

“Alkylalkoxy” refers to the group “-alklene-O-alkyl” wherein alkyleneand alkoxy are as defined herein. Such groups include, by way ofexample, methyl methoxy (—CH₂OCH₃), ethyl methoxy (—CH₂CH₂OCH₃),n-propyl-iso-propoxy (—CH₂CH₂CH₂OCH(CH₃)₂), methyl-tert-butoxy(—CH₂—O—C(CH₃)₃) and the like.

“Alkylthioalkoxy” refers to the group “-alkylene-S-alkyl” whereinalkylene and alkoxy are as defined herein. Such groups include, by wayof example, methylthiomethoxy (—CH₂SCH₃), ethylthiomethoxy(—CH₂CH₂SCH₃), n-propyl-iso-thiopropoxy (—CH₂CH₂CH₂SCH(CH₃)₂),methyl-tert-thiobutoxy (—CH₂SC(CH₃)₃) and the like.

“Alkenyl” refers to alkenyl groups preferably having from 2 to 10 carbonatoms and more preferably 2 to 6 carbon atoms and having at least 1 andpreferably from 1-2 sites of alkenyl unsaturation. Preferred alkenylgroups include ethenyl (—CH═CH₂), n-propenyl (—CH₂CH═CH₂), iso-propenyl(—C(CH₃)═CH₂), but-2-enyl (—CH₂CH═CHCH₃) and the like.

“Alkynyl” refers to alkynyl groups preferably having from 2 to 10 carbonatoms and more preferably 2 to 6 carbon atoms and having at least 1 andpreferably from 1-2 sites of alkynyl unsaturation. Preferred alkynylgroups include ethynyl (—C≡CH), propargyl (—CH₂C≡CH) and the like.

“Acyl” refers to the groups alkyl-C(O)—, aryl-C(O)—, andheteroaryl-C(O)— where alkyl, aryl and heteroaryl are as defined herein.

“Acylamino” refers to the group —C(O)NRR where each R is independentlyhydrogen or alkyl where alkyl is as defined herein.

“Aminoacyl” refers to the group —NRC(O)R where each R is independentlyhydrogen or alkyl where alkyl is as defined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, aryl-C(O)O—,heteroaryl-C(O)O—, and heterocyclic-C(O)O— where alkyl, aryl, heteroaryland heterocyclic are as defined herein.

“Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl). Preferred aryls includephenyl, naphthyl and the like.

Unless otherwise constrained by the definition for the aryl substituent,such aryl groups can optionally be substituted with from 1 to 3substituents selected from the group consisting of hydroxy, acyl,acyloxy, alkyl, alkoxy, alkenyl, alkynyl, amino, aminoacyl, aryl,aryloxy, carboxyl, alkoxycarbonyl, acylamino, cyano, halo, nitro,heteroaryl, trihalomethyl and the like. Preferred substituents includealkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.

“Aryloxy” refers to the group aryl-O— wherein the aryl group is asdefined above including optionally substituted aryl groups as alsodefined above.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving a single cyclic ring or multiple condensed rings which can beoptionally substituted with from 1 to 3 alkyl groups. Such cycloalkylgroups include, by way of example, single ring structures such ascyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl,2-methylcyclopentyl, 2-methylcyclooctyl, and the like, or multiple ringstructures such as adamantanyl, and the like.

“Cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 8 carbonatoms having a single cyclic ring and at least one point of internalunsaturation which can be optionally substituted with from 1 to 3 alkylgroups. Examples of suitable cycloalkenyl groups include, for instance,cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo andpreferably is either chloro or fluoro.

“Heteroaryl” refers to a monovalent aromatic group of from 2 to 8 carbonatoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfurwithin the ring.

Unless otherwise constrained by the definition for the heteroarylsubstituent, such heteroaryl groups can be optionally substituted with 1to 3 substituents selected from the group consisting of alkyl, alkoxy,aryl, aryloxy, halo, nitro, heteroaryl, thioalkoxy, thioaryloxy and thelike. Such heteroaryl groups can have a single ring (e.g., pyridyl orfuryl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).Preferred heteroaryls include pyridyl, pyrrolyl and furyl.

“Heterocycle” or “heterocyclic” refers to a monovalent saturated orunsaturated group having a single ring or multiple condensed rings, from1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen,sulfur or oxygen within the ring.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 3 substituents selected from the group consisting of alkyl, alkoxy,aryl, aryloxy, halo, nitro, heteroaryl, thioalkoxy, thioaryloxy and thelike. Such heterocyclic groups can have a single ring (e.g., piperidinylor tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl,dihydrobenzoftu or quinuclidinyl). Preferred heterocycles includepiperidinyl, pyrrolidinyl and tetrahydrofiiryl.

Examples of heterocycles and heteroaryls include, but are not limitedto, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine,phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine,phenothiazine, imidazolidine, imidazoline, piperidine, piperazine,pyrrolidine, indoline and the like.

In the compounds of formula I, R^(b) and R^(c) can be fused to form aheteroaryl or heterocyclic ring with the phenyl ring. Fusion in thismanner results in a fused bicyclic ring structure of the formula:

where R^(b′) is as defined above and A is the fused heteroaryl orheterocyclic group containing from 3 to 8 atoms of which from 1 to 3 areheteroatoms independently selected from the group consisting of oxygen,nitrogen and sulfur wherein the two atoms of the phenyl ring areincluded in the total atoms present in the heteroaryl or heterocyclicgroup. Examples of such fused ring systems include, for instance,indol-5-yl, indol-6-yl, thionaphthen-5-yl, thionaphthen-6-yl,isothionaphthen-5-yl, isothionaphthen-6-yl, indoxazin-5-yl,indoxazin-6-yl, benzoxazol-5-yl, benzoxazol-6-yl, anthranil-5-yl,anthranil-6-yl, quinolin-6-yl, quinolin-7-yl, isoquinolin-6-yl,isoquinolin-7-yl, cinnolin-6-yl, cinnolin-7-yl, quinazolin-6-yl,quinazolin-7-yl, benzofuran-5-yl, benzofuran-6-yl, isobenzofuran-5-yl,isobenzofuran-6-yl, and the like.

“Thiol” refers to the group —SH.

“Thioalkoxy” refers to the group —S-alkyl.

“Thioaryloxy” refers to the group aryl-S— wherein the aryl group is asdefined above including optionally substituted aryl groups as alsodefined above.

“Thioheteroaryloxy” refers to the group heteroaryl-S— wherein theheteroaryl group is as defined above including optionally substitutedaryl groups as also defined above.

“Pharmaceutically acceptable salt” refers to pharmaceutically acceptablesalts of a compound of Formula I which salts are derived from a varietyof organic and inorganic counter ions well known in the art and include,by way of example only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate and the like.

Compound Preparation

The compounds of formula I above are readily prepared via severaldivergent synthetic routes with the particular route selected relativeto the ease of compound preparation, the commercial availability ofstarting materials, and the like.

A first synthetic method involves conventional coupling of a halo aceticacid with a primary amine to form the amino acid followed byconventional esterification as shown in reaction (1) below:

wherein R¹, R², R³ are as defined above and X is a halo group such aschloro or bromo. Alternatively, leaving groups other than halo may beemployed such as triflate, tosylate, mesylate and the like.Additionally, a suitable ester of 1 may be employed in this reaction.

The first step of reaction (1) involves coupling of a suitablehaloacetic acid derivative 1 with a primary aryl/heteroarylamine 2 underconditions which provide for amino acid 3. This reaction is describedby, for example, Yates, et al.¹⁰ and proceeds by combining approximatelystoichiometric equivalents of haloacetic acid 1 with primaryaryl/heteroarylamine 2 in a suitable inert diluent such as water,dimethylsulfoxide (DMSO) and the like. The reaction employs an excess ofa suitable base such as sodium bicarbonate, sodium hydroxide, etc. toscavenge the acid generated by the reaction. The reaction is preferablyconducted at from about 25° C. to about 100° C. until reactioncompletion which typically occurs within 1 to about 24 hours. Thisreaction is further described in U.S. Pat. No. 3,598,859, which isincorporated herein by reference in its entirety. Upon reactioncompletion, N-aryl/N-heteroaryl amino acid 3 is recovered byconventional methods including precipitation, chromatography, filtrationand the like.

N-aryl/N-heteroaryl amino acid 3 is next esterified with alcohol 4 byconventional esterificafion conditions to provide for the esterifiedN-aryl/N-heteroaryl amino acid 5 which is a compound of formula I. Forexample, esterification procedures for R³ groups containing an estergroup can be achieved by using the methods of Losse, et al.¹¹ Ifdesired, the esterification reaction can optionally be conducted onhaloacetic acid 1 prior to amination with aryl/heteroarylamine 2.

In reaction (1), each of the reagents (haloacetic acid 1, primaryaryl/heteroarylamine 2 and alcohol 3) are well known in the art with aplurality of each being commercially available.

In an alternative embodiment, the R¹ group can be coupled to an alanineester (or other suitable amino acid ester) by conventional N-arylation.For example, a stoichiometric equivalent or slight excess of the aminoacid ester can be dissolved in a suitable diluent such as DMSO andcoupled with a haloaryl compound, X—R¹ where X is a halo group such asfluoro, chloro or bromo and R¹ is as defined above. The reaction isconducted in the presence of an excess of base such as sodium hydroxideto scavenge the acid generated by the reaction. The reaction typicallyproceeds at from 15° C. to about 250° C. and is complete in about 1 to24 hours. Upon reaction completion, N-aryl amino acid ester is recoveredby conventional methods including chromatography, filtration and thelike.

In still another alternative embodiment, the esterified amino acids offormula I above can be prepared by reductive amination of a2-oxocarboxylic acid ester (such as a pyruvate ester) ester in themanner illustrated in Reaction (2) below:

wherein R¹, R², R³ are as defined above.

In reaction (2), approximately stoichiometric equivalents of pyruvateester 6 and arylamine 2 are combined in an inert diluent such asmethanol, ethanol and the like and the reaction solution treated underconditions which provide for imine formation (not shown). The imineformed is then reduced under conventional conditions by a suitablereducing agent such as sodium cyanoborohydride, H₂/palladium on carbonand the like to form the N-aryl amino acid ester 5. In a particularlypreferred embodiment, the reducing agent is H₂/palladium on carbon whichis incorporated into the initial reaction medium which permits iminereduction in situ in a one pot procedure to provide for the N-aryl aminoacid ester 5.

The reaction is preferably conducted at from about 20° C. to about 80°C. at a pressure of from 1 to 10 atmospheres until reaction completionwhich typically occurs within 1 to about 24 hours. Upon reactioncompletion, N-aryl amino acid ester 5 is recovered by conventionalmethods including chromatography, filtration and the like.

A further embodiment for preparing the compounds of formula I aboveincludes aromatic nucleophilic substitution of fluorobenzenes by theamine group of an amino acid as set forth in the Examples below.

In still a further embodiment, conventional transesterificationtechniques can be used to prepare a variety of different ester groups onthe N-aryl amino acid esters 5. Numerous techniques are known in the artto effect transesterification and each technique merely replaces the—OR³ group on the ester of the N-aryl amino acid ester 5 with adifferent —OR³ group derived from the corresponding alcohol (i.e., HOR³)and, in some cases, a catalyst such as titanium (IV) iso-propoxide isused to facilitate reaction completion. In one technique, the alcoholHOR³ is first treated with sodium hydride in a suitable diluent such astoluene to form the corresponding Na⁺ OR³ which is then employed toeffect transesterification with the N-aryl amino acid ester 5. Theefficiency of this technique makes it particularly useful with highboiling and/or expensive alcohols.

In another transesterification technique, the N-aryl amino acid ester 5to be transesterified is placed in a large excess of the alcohol whicheffects transesterification. A catalytic amount of sodium hydride isthen added and the reaction proceeds quickly under conventionalconditions to provide the desired transesterified product. Because thisprotocol requires the use of a large excess of alcohol, this procedureis particularly useful when the alcohol is inexpensive.

Transesterification provides a facile means to provide for amultiplicity of R³ substituents on the compounds of formula I above. Inall cases, the alcohols employed to effect transesterification are wellknown in the art with a significant number being commercially available.

Other methods for preparing the esters of this invention include, by wayof example, first hydrolyzing the ester to the free acid followed byO-alkylation with a halo-R³ group in the presence of a base such aspotassium carbonate.

Still other methods for the preparation of esters are provided in theexamples below.

Methods for the preparation of O-acyloxime esters includetransesterification of the trichlorophenyl ester of a carboxylic acidwith an oxime, and coupling of a carboxylic acid and an oxime using acarbodiimide coupling reagent. Similarly, methods for the preparation ofpyrrole amides include conventional amidation techniques of thecorresponding acid and pyrrole.

In these synthetic methods, the starting materials can contain a chiralcenter (e.g., alanine) and, when a racemic starting material isemployed, the resulting product is a mixture of R,S enantiomers.Alternatively, a chiral isomer of the starting material can be employedand, if the reaction protocol employed does not racemize this startingmaterial, a chiral product is obtained. Such reaction protocols caninvolve inversion of the chiral center during synthesis.

Accordingly, unless otherwise indicated, the products of this inventionare a mixture of R,S enantiomers. Preferably, however, when a chiralproduct is desired, the chiral product corresponds to the L-amino acidderivative. Alternatively, chiral products can be obtained viapurification techniques which separates enantiomers from a R,S mixtureto provide for one or the other stereoisomer. Such techniques are wellknown in the art.

Pharmaceutical Formulations

When employed as pharmaceuticals, the compounds of formula I are usuallyadministered in the form of pharmaceutical compositions. These compoundscan be administered by a variety of routes including oral, rectal,transdermal, subcutaneous, intravenous, intramuscular, and intranasal.These compounds are effective as both injectable and oral compositions.Such compositions are prepared in a manner well known in thepharmaceutical art and comprise at least one active compound.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds of formula Iabove associated with pharmaceutically acceptable carriers. In makingthe compositions of this invention, the active ingredient is usuallymixed with an excipient, diluted by an excipient or enclosed within sucha carrier which can be in the form of a capsule, sachet, paper or othercontainer. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activecompound to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active compound is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 5 to about 100 mg, more usually about 10 toabout 30 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient. Preferably, the compound of formula I above is employed at nomore than about 20 weight percent of the pharmaceutical composition,more preferably no more than about 15 weight percent, with the balancebeing pharmaceutically inert carrier(s).

The active compound is effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It, willbe understood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

The following formulation examples illustrate representativepharmaceutical compositions of the present invention.

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below:

Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose,microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Ingredient Weight % Active Ingredient 5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows:

Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mg Starch 45.0 mgMicrocrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0 mg (as 10%solution in sterile water) Sodium carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc 1.0 mg Total 120 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

Formulation Example 5

Capsules, each containing 40 mg of medicament are made as follows:

Quantity Ingredient (mg/capsule) Active Ingredient 40.0 mg Starch 109.0mg Magnesium stearate 1.0 mg Total 150.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 150 mg quantities.

Formulation Example 6

Suppositories, each containing 25 mg of active ingredient are made asfollows:

Ingredient Amount Active Ingredient 25 mg Saturated fatty acidglycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 7

Suspensions, each containing 50 mg of medicament per 5.0 mL dose aremade as follows:

Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodiumcarboxymethyl cellulose (11%) 50.0 mg Microcrystalline cellulose (89%)Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purifiedwater to 5.0 mL

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

Formulation Example 8

Quantity Ingredient (mg/capsule) Active Ingredient 15.0 mg Starch 407.0mg Magnesium stearate 3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 425.0 mg quantities.

Formulation Example 9

A subcutaneous formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 5.0 mg Corn Oil 1.0 mL

Formulation Example 10

A topical formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 1-10 g Emulsifying Wax 30 g LiquidParaffin 20 g White Soft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

Frequently, it will be desirable or necessary to introduce thepharmaceutical composition to the brain, either directly or indirectly.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system used for the transport ofbiological factors to specific anatomical regions of the body isdescribed in U.S. Pat. No. 5,011,472 which is herein incorporated byreference.

Indirect techniques, which are generally preferred, usually involveformulating the compositions to provide for drug latentiation by theconversion of hydrophilic drugs into lipid-soluble drugs. Latentiationis generally achieved through blocking of the hydroxy, carbonyl,sulfate, and primary amine groups present on the drug to render the drugmore lipid soluble and amenable to transportation across the blood-brainbarrier. Alternatively, the delivery of hydrophilic drugs may beenhanced by intra-arterial infusion of hypertonic solutions which cantransiently open the blood-brain barrier.

Other suitable formulations for use in the present invention can befound in Remington's Phannaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985).

Utility

The compounds and pharmaceutical compositions of the invention areuseful in inhibiting β-amyloid peptide release and/or its synthesis,and, accordingly, have utility in treating Alzheimer's disease inmammals including humans.

As noted above, the compounds described herein are suitable for use in avariety of drug delivery systems described above. Additionally, in orderto enhance the in vivo serum half-life of the administered compound, thecompounds may be encapsulated, introduced into the lumen of liposomes,prepared as a colloid, or other conventional techniques may be employedwhich provide an extended serum half-life of the compounds. A variety ofmethods are available for preparing liposomes, as described in, e.g.,Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028 each ofwhich is incorporated herein by reference.

The amount of compound administered to the patient will vary dependingupon what is being administered, the purpose of the administration, suchas prophylaxis or therapy, the state of the patient, the manner ofadministration, and the like. In therapeutic applications, compositionsare administered to a patient already suffering from AD in an amountsufficient to at least partially arrest further onset of the symptoms ofthe disease and its complications. An amount adequate to accomplish thisis defined as “therapeutically effective dose.” Amounts effective forthis use will depend on the judgment of the attending cliniciandepending upon factors such as the degree or severity of AD in thepatient, the age, weight and general condition of the patient, and thelike. Preferably, for use as therapeutics, the compounds describedherein are administered at dosages ranging from about 0.1 to about 500mg/kg/day.

In prophylactic applications, compositions are administered to a patientat risk of developing AD (determined for example by genetic screening orfamilial trait) in an amount sufficient to inhibit the onset of symptomsof the disease. An amount adequate to accomplish this is defined as“prophylactically effective dose.” Amounts effective for this use willdepend on the judgment of the attending clinician depending upon factorssuch as the age, weight and general condition of the patient, and thelike. Preferably, for use as prophylactics, the compounds describedherein are administered at dosages ranging from about 0.1 to about 500mg/kg/day.

As noted above, the compounds administered to a patient are in the formof pharmaceutical compositions described above. These compositions maybe sterilized by conventional sterilization techniques, or may besterile filtered. When aqueous solutions are employed, these may bepackaged for use as is, or lyophilized, the lyophilized preparationbeing combined with a sterile aqueous carrier prior to administration.The pH of the compound preparations typically will be between 3 and 11,more preferably from 5-9 and most preferably from 7 and 8. It will beunderstood that use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of pharmaceutical salts.

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention. Unless otherwise stated, alltemperatures are in degrees Celsius.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning.

BOC = tert-butoxycarbonyl bd = broad doublet bs = broad singlet cc =cubic centimeter d = doublet dd = doublet of doublets DMF =dimethylformamide DMSO = dimethyl sulfoxide EDC =1-(3-dimethyaminopropyl)-ethylcarbodiimide hydrochloride EDTA = ethylenediamine tetraacetic acid eq. = equivalents ether = diethyl ether g =grams hept. = heptuplet m = multiplet M = molar max = maximum mg =milligram min. = minutes mL = milliliter mM = millimolar mmol =millimole N = normal ng = nanogram nm = nanometers OD = optical densitypg = picogram pM = picomolar φ = phenyl psi = pounds per square inch q =quartet quint. = quintuplet rpm = rotations per minute s = singlet sept= septuplet t = triplet THF = tetrahydrofuran tlc = thin layerchromatography μL = microliter UV = ultraviolet w/v = weight to volume

Additionally, the term “Aldrich” indicates that the compound or reagentused in the following procedures is commercially available from AldrichChemical Company, Inc., 1001 West Saint Paul Avenue, Milwaukee, Wis.53233 USA; the term “Fluka” indicates the compound or reagent iscommercially available from Fluka Chemical Corp., 980 South 2nd Street,Ronkonkoma, N.Y. 11779 USA; the term “Lancaster” indicates the compoundor reagent is commercially available from Lancaster Synthesis, Inc.,P.O. Box 100, Windham, N.H. 03087 USA; and the term “Sigma” indicatesthe compound or reagent is commercially available from Sigma, P.O. Box14508, St. Louis, Mo. 63178 USA.

In the examples below, all temperatures are in degrees Celsius (unlessotherwise indicated) and the following general procedures were used toprepare the compounds as indicated.

General Procedure A Reductive Amination

To a solution of the arylamine in ethanol in a hydrogenation flask wasadded 1 equivalent of the 2-oxocarboxylic acid ester (e.g., pyruvateester), followed by 10% palladium on carbon (25 weight percent based onthe arylamine). The reaction was hydrogenated at 20 psi H₂ on a Parrshaker until complete reaction was indicated by tlc (30 minutes to 16hours). The reaction mixture was then filtered through a pad of Celite545 (available from Aldrich Chemical Company, Inc.) and stripped free ofsolvent on a rotary evaporator. The crude product residue was thenfurther purified via chromatography.

General Procedure B First Transesterification Technique

A solution of 1-5 equivalents of the desired alcohol was added to 1equivalent of sodium hydride in toluene. After off-gassing had ceased,the compound to be transesterified, dissolved in toluene, was added.After 0.5 hours, the reaction was either heated to 40° C. and placedunder house vacuum (˜20 mmHg), or nitrogen was bubbled through thesolution while it was heated at 90° C. The reaction was followed by tlc,and when the reaction was complete the solution was cooled and quenchedwith water or 1M HCl, and in smaller scale reactions diluted with ethylacetate. The organic phase was extracted with saturated aqueous NaHCO₃,then washed with saturated aqueous NaCl and dried over MgSO₄. Thesolution was stripped free of solvent on a rotary evaporator, and thecrude product residue was then further purified by chromatography.Alternatively, the reaction mixture was worked-up by evaporation of thesolvents and direct chromatography of the crude mixture.

This procedure is particularly useful in the case of costly and/or highboiling alcohols.

General Procedure C Second Transesterification Technique

The compound to be transesterified was placed in a large excess of thedesired alcohol. A catalytic amount of dry NaH was added, and thereaction was followed by tlc until the presence of starting material wasno longer detected. The reaction was quenched with a few milliliters of1N HCl, and after a few minutes of stirring saturated aqueous NaHCO₃ wasadded. The organic phase was washed with saturated aqueous NaCl anddried over MgSO₄. The solution was stripped free of solvent on a rotaryevaporator, and the crude product residue was then further purified bychromatography.

General Procedure D Third Transesterification Technique

The compound to be transesterified was placed in a large excess of thedesired alcohol. A catalytic amount of dry NaH was added, and thereaction was followed by tlc until the presence of starting material wasno longer detected. The reaction was quenched with a few milliliters of1N HCl, and after a few minutes of stirring saturated aqueous NaHCO₃ wasadded. The volume of the reaction mixture was reduced on a rotaryevaporator until the excess alcohol was removed and then the remainingresidue was taken up in ethyl acetate and additional water was added.The organic phase was washed with saturated aqueous NaCl and dried overMgSO₄. The solution was stripped free of solvent on a rotary evaporator,and the crude product residue was then further purified bychromatography.

This procedure is particularly employed in the case of low boiling,inexpensive alcohols, miscible with water.

General Procedure E O-Alkylation Technique

To a carboxylic acid compound (prepared, for example, by reductiveamination via General Procedure A to provide for the N-aryl amino acidester, followed by hydrolysis via Procedure F) in DMF was added 1.5equivalents K₂CO₃, followed by 1 equivalent of alkylating agent (e.g.,tert-butyl bromoacetate). The reaction was stirred at room temperaturefor 2 hours, then was quenched with water and extracted into ethylacetate. The organic phase was washed with saturated aqueous NaHCO₃,water, and saturated aqueous NaCl, and was then dried over MgSO₄. Thesolution was stripped free of solvent on a rotary evaporator to yieldthe crude product.

General Procedure F Ester Hydrolysis to Free Acid

To a carboxylic ester compound (prepared, for example, by reductiveamination via General Procedure A to provide for the N-aryl amino acidester) in a 1:1 mixture of CH₃OH/H₂O was added 2-5 equivalents of K₂CO₃.The mixture was heated to 50° C. for 0.5 to 1.5 hours until tlc showedcomplete reaction. The reaction was cooled to room temperature and themethanol was removed on a rotary evaporator. The pH of the remainingaqueous solution was adjusted to ˜2, and ethyl acetate was added toextract the product. The organic phase was then washed with saturatedaqueous NaCl and dried over MgSO₄. The solution was stripped free ofsolvent on a rotary evaporator to yield the crude product.

General Procedure G N-Heteroarylation of Alanine

A solution of 1.1 equivalents of L-alanine and 2 equivalents NaOH inDMSO was stirred at room temperature for 1 hour, then 1 equivalent of2-chlorobenzothiazole was added. The mixture was heated to 100° C. for 4hours, then cooled to room temperature and poured onto ice. The pH ofthe resulting aqueous solution was adjusted to ˜2, and the precipitatedsolid was removed by filtration. This solid was then dissolved in 1NNaOH and the resulting solution was filtered through a pad of Celite545. The pH of the filtrate was adjusted to ˜2, and the whiteprecipitate was removed by filtration and washed with water to yield thecrude product.

General Procedure H EDC Coupling

To a 1:1 mixture of the desired acid and alcohol in CH₂Cl₂ at 0° C. wasadded 1.5 equivalents triethylamine, followed by 2.0 equivalentshydroxybenzotriazole monohydrate, then 1.25 equivalents ofethyl-3-(3-dimethylamino)-propyl carbodiimideHCl (EDC). The reaction wasstirred overnight at room temperature, then transferred to a separatoryfunnel and washed with water, saturated aqueous NaHCO₃, 1N HCl, andsaturated aqueous NaCl, and was then dried over MgSO₄. The solution wasstripped free of solvent on a rotary evaporator to yield the crudeproduct.

General Procedure I Oxime or Amine Coupling Technique

The trichlorophenyl ester (1 eq) of a carboxylic acid was stirred in DMFor THF. The oxime or amine (1.2 eq) was added and the mixture wasstirred at ambient temperature for 1-4 hours. In cases where thehydrochloride salt form of an amine was used, a suitable base such asN,N-diisopropylethylamine (1.2 eq) was also added. The resulting mixturewas concentrated under reduced pressure to yield a crude product whichwas used without purification or was purified by silica gelchromatography and/or crystallization.

General Procedure J Alkylation Technique

The amine (1 eq), the α-bromo ester (1.1 eq) and a suitable base (suchas triethylamine) (2 eq) were stirred in chloroform. The resultingsolution was heated at reflux for 4-12 hours. After cooling, the mixturewas diluted with chloroform and washed with water. The organic portionwas dried (sodium sulfate) and concentrated under reduced pressure. Thecrude product was purified by silica gel chromatography.

General Procedure K Oxime or Alcohol Coupling Technique

The carboxylic acid (1 eq) was stirred in a suitable solvent (such asTHF, dioxane or DMF). An alcohol or oxime (1-5 eq) was added. EDC Shydrochloride (1.2 eq) and hydroxybenzotriazole hydrate (1 eq) wereadded. A suitable base (such as 4-methylmorpholine or triethylamine)(0-1 eq) was added. A catalytic amount (0.1 eq) of4-dimethylaminopyridine was added. The mixture was stirred at ambienttemperature and under a dry atmosphere of nitrogen. After 20 hours, themixture was concentrated under reduced pressure. The resultingconcentrate was partitioned between ethyl acetate and water. The organicportion was separated and washed with aqueous sodium bicarbonate andbrine. The organic portion was dried (sodium sulfate) and concentratedunder reduced pressure. The crude product was used without purificationor was purified by silica gel chromatography and/or crystallization.

General Procedure L EDC Coupling

The carboxylic acid was dissolved in methylene chloride. The amino acid(1 eq.), N-methylmorpholine (5 eq.) and hydroxybenzotriazole monohydrate(1.2 eq.) were added in sequence. A cooling bath was applied to theround bottomed flask until the solution reached 0° C. At that time, 1.2eq. of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC)was added. The solution was allowed to stir overnight and come to roomtemperature under nitrogen pressure. The reaction mixture was worked upby washing the organic phase with saturated aqueous sodium carbonate,0.1M citric acid, and brine before drying with sodium sulfate. Thesolvents were then removed to yield crude product. Pure products wereobtained by flash chromatography in an appropriate solvent.

General Procedure M Triflate Displacement

To a 0° C. solution of iso-butyl R-(+)-lactate in CH₂Cl₂ was added 1.1equivalents of trifluoromethanesulfonic anhydride. After stirring atroom temperature for 20 min, 1.1 equivalents of 2,6-lutidine was addedand stiring was continued for 10 min. This solution was then transferredto a flask containing 1 equivalent the arylamine and 1 equivalentN,N-diisopropylethylamine in CH₂Cl₂ or CH₃NO₂ at 0° C. The reaction washeld overnight at room temperature and then stripped free of solvent ona rotary evaporator. The residue was dissolved in ethyl acetate, washedwith 5% citric acid, followed by saturated aqueous NaCl, dried overmagnesium sulfate or sodium sulfate and then the solution was strippedfree of solvent on a rotary evaporator to yield the crude product, whichwas then purified by chromatography.

General Procedure N BOC Removal

The BOC-protected compound was added to a 1:1 mixture of CH₂Cl₂ andtrifluoroacetic acid, and was stirred until tlc indicated completeconversion, typically 2 h. The solution was then stripped to dryness andthe residue was taken up in ethyl acetate and extracted with dilute HCl.The acid reaction was neutralized and extracted with ethyl acetate. Theorganic phase was washed with saturated aqueous NaCl and dried overMgSO₄. The solution was stripped free of solvent on a rotary evaporatorto yield the product.

General Procedure O Synthesis of Pyruvate Esters

To a mixture of pyruvic acid (8.8 g, 0.1 mol) (Aldrich) in 100 mL ofbenzene was added isobutanol (14.82 g, 0.2 mol) and a catalytic amountof p-toluenesulfonic acid. The mixture was then refluxed using a DeanStark apparatus. After 4 hours, the reaction appeared to be completewith the isolation of 1.8 g (0.1 mol) of water. The benzene andiso-butanol were removed on a rotary evaporator. The residue (14 g, 0.1mol), which was primarily the pyruvate iso-butyl ester by nmr [¹H-Nmr(CDCl₃): δ=4.0 (d, 2H), 2.5 (s, 3H), 2.0 (m, 1H), 1.0 (d, 6H)], was usedwithout further purification. By substituting other alcohols in place ofisobutanol (e.g., ethanol, isopropanol, n-butanol, benzyl alcohol andthe like), other esters of pyruvic acid can be prepared in a similarmanner.

General Procedure P Aromatic Nucleophilic Substitution of Fluorobenzenes

A mixture of 1.82 g (10 mmol) of D,L-alanine iso-butyl esterhydrochloride, the fluorobenzene (10 mmol) and 3 g of anhydrouspotassium carbonate in 10 mL of DMSO was stirred at 120° C. for 2-5hours. The reaction mixture was then cooled to room temperature anddiluted with 100 mL of ethyl acetate. The ethyl acetate extract waswashed with water (3×), dried over MgSO₄ and evaporated to dryness toafford the crude product, which was further purified by columnchromatography.

General Procedure Q Fourth Transesterification Technique

The ester to be transesterified was dissolved in a large excess of thealcohol and 0.3 equivalents of titanium(IV) isopropoxide (Aldrich) wasadded. The reaction was followed by tlc until complete and then thevolatiles were removed at reduced pressure. The resulting crude materialwas then chromatographed to obtain the desired product.

General Procedure R Synthesis on N-BOC Anilines

To a solution of the aniline in THF was added dropwise 1 equivalent ofdi-tert-butyl dicarbonate (Aldrich) in THF and then 1.5 equivalents of10N aqueous sodium hydroxide at 0° C. After stirring at room temperaturefor 16 hours, or heating at 80° C. for 3 hours, if needed, the reactionmixture was diluted with ether and washed with NaHCO₃, brine, dried oversodium sulfate and potassium carbonate, concentrated at reduced pressureand chromatographed to afford the N-BOC aniline.

General Procedure S Oxime Ester Formation

The trichlorophenyl ester (1 eq.) was stirred in DMF or THF. The oxime(1.2 eq.) was added and the mixture was stirred at ambient temperaturefor 1 to 4 hours. The resulting mixture was concentrated under reducedpressure and the residue was purified by silica gel chromatographyand/or crystallization.

Example A Synthesis of D,Lalanine iso-butyl ester hydrochloride

A mixture of 35.64 g (0.4 mol) of D,L-alanine (Aldrich), 44 mL (0.6 mol)of thionyl chloride (Aldrich) and 200 mL of iso-butanol was refluxed for1.5 hours. The volatiles were removed at reduced pressure at 90° C.under reduced pressure to give the title compound as an oil, which wasused without further purification.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=8.72 (br s, 3H), 4.27 (q, J=7.4 Hz, 1H), 3.95 (m, 2H),1.96 (s, 1H), 1.73 (d, J=7.2 Hz, 3H), 0.92 (d, J=6.7 Hz, 6H).

¹³C-nmr (CDCl₃): δ=170.0, 72.2, 49.2, 27.5, 18.9, 16.1.

Example B Synthesis of N-(3,4dichlorophenyl)alanine

Using the procedure set forth in U.S. Pat. No. 3,598,859, the disclosureof which is incorporated herein by reference in its entirety,N-(3,4-dichlorophenyl)alanine was prepared. Specifically, to a solutionof 3,4-dichloroaniline (1 equivalent) (Aldrich) in isopropanol (about500 mL per mole of 3,4-dichloroaniline) is added water (about 0.06 mLper mL of isopropanol) and 2-chloropropionic acid (2 equivalents)(Aldrich). This mixture is warmed to 40° C. and sodium bicarbonate (0.25equivalents) is added in successive portions before heating under refluxfor 4-5 days. After cooling, the reaction mixture is poured into waterand the unreacted 3,4-dichloroaniline is removed by filtration. Thefiltrate is acidified to pH 3-4 with concentrated hydrochloric acid andthe resultant precipitate is filtered, washed and dried to yield thetitle compound, m.p.=148-149° C.

Alternatively, following General Procedure F above and usingN-(3,4-dichlorophenyl)alanine ethyl ester (from Example 1 below), thetitle compound was prepared.

Example C Synthesis of N-(3,5-difluorophenyl)alanine

Using the procedure set forth in U.S. Pat. No. 3,598,859,N-(3,5-difluorophenyl)alanine was prepared using 3,5-difluoroaniline(Aldrich) and 2-chloropropionic acid (Aldrich).

Example D Synthesis of Iso-butyl 2-bromopropionate

To a mixture of iso-butanol and 1.0 equivalent of pyridine in drydiethyl ether was added dropwise 1.3 equivalents of 2-bromopropionylbromide at 0° C. After stirring at room temperature for 16 hours, thereaction was diluted with diethyl ether, washed with 1N HCl, water,aqueous NaHCO₃, brine and dried over magnesium sulfate or sodiumsulfate. Removal of the solvents at reduced pressure gave the titlecompound as a clear oil.

Example E Synthesis of N-(2-naphthyl)alanine 2,4,5-trichlorophenyl ester

N-(2-Naphthyl)alanine methyl ester (5.0 g, 20.6 mmol) (from Example 44below) was dissolved in dioxane (100 mL). NaOH (30 mL, 1N) was added andthe resulting solution was stirred for 1 hour. The reaction mixture wasconcentrated under reduced pressure. The resulting solid was dissolvedin water and the aqueous mixture was washed with ether. The aqueousportion was adjusted to pH 3 with 1N HCl and extracted with ethylacetate. The organic extracts were dried over magnesium sulfate orsodium sulfate and concentrated under reduced pressure to yield a whitesolid (4.35 g, 98%).

The resulting solid (4.35 g, 20 mmol) was dissolved in dichloromethane(300 mL). 2,4,5-Trichlorophenol (4.9 g, 25 mmol) (Aldrich) was addedfollowed by dicyclohexylcarbodiimide (25 mL, 1M in dichloromethane)(Aldrich). After stirring for 18 hours, the mixture was filtered andconcentrated to provide an oil which was purified by chromatography onsilica gel using chloroform as the eluant (R_(f)=0.6). The titlecompound was obtained as a thick oil which slowly crystallized.

Example 1 Synthesis of N-(3,4-dichlorophenyl)alanine ethyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and ethyl pyruvate (Aldrich), the title compound was preparedas an oil. The reaction was monitored by tlc on silica gel (R_(f)=0.4 in25% EtOAc/hexanes) and purification was by preparative platechromatography (silica gel using 25 % EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.2 (d, 1H); 6.7 (d, 1H,); 6.4 (dd, 1H); 4.30 (bs,1H); 4.2 (q, 2H); 4.1 (q, 1H); 1.5 (d, 3H); 1.3 (t, 3H).

¹³C-nmr (CDCl₃): δ=175; 146.7; 133; 131; 121; 114.9; 112.6; 72.0; 52.4;28.3; 19.5.

C₁₁H₁₃Cl₂NO₂ (MW=262.14).

Example 2 Synthesis of N-(3-trifluoromethyl-4-chlorophenyl)alanine ethylester

Following General Procedure A above and using4-chloro-3-(trifluoromethyl)aniline (Aldrich) and ethyl pyruvate(Aldrich), the title compound was prepared.

Analysis: Calc.: C, 48.74; H, 4.43; N, 4.74. Found: C, 48.48; H, 4.54;N, 4.94.

C₁₂H₁₃F₃ClNO₂ (MW=295.69); mass spectroscopy (MH⁺) 295.

Example 3 Synthesis of N-(3,5-dichlorophenyl)alanine ethyl ester

Following General Procedure A above and using 3,5-dichloroaniline(Aldrich) and ethyl pyruvate (Aldrich), the title compound was prepared.

Analysis: Calc.: C, 50.40; H, 5.00; N, 5.34. Found: C, 50.50; H, 5.06;N, 5.25.

C₁₁H₁₃Cl₂NO₂ (MW=262.14); mass spectroscopy (MH⁺) NA.

Example 4 Synthesis of N-(3,4-difluorophenyl)alanine ethyl ester

Following General Procedure A above and using 3,4-difluoroaniline(Aldrich) and ethyl pyruvate (Aldrich), the title compound was prepared.The reaction was monitored by tlc on silica gel (Rf=0.4 in 25%EtOAc/hexanes) and purification was by preparative plate chromatography(silica gel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.4 (m, 1H), 6.8 (d, 1H), 6.5 (m, 1H), 4.30 (bs, 1H),4.2 (q, 2H), 4.1 (q, 1H), 1.5 (d, 3H), 1.3 (t, 3H).

¹³C-nmr (CDCl₃): δ=175, 146.7, 135, 132, 125, 116, 113, 72, 52, 28, 19.

C₁₁H₁₃F₂NO₂ (MW=229.23); mass spectroscopy (MH⁺) 230.

Example 5 Synthesis of N-(3,4-dichlorophenyl)alanine benzyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and benzyl pyruvate (prepared by following General Procedure Oabove using benzyl alcohol in place of isobutanol), the title compoundwas prepared as an oil. The reaction was monitored by tlc on silica gel(Rf=0.4 in 25% EtOAc/hexanes) and purification was by preparative platechromatography (silica gel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.18 (d, 1H); 7.0 (m, 5H); 6.6 (d, 1H,); 6.4 (dd, 1H);5.1 (s, 2H); 4.30 (bs, 1H); 4.08 (q, 1H); 1.94 (m, 1H); 1.47 (d, 3H);0.91 (d, 6H).

¹³C-nmr (CDCl₃): δ=174.5; 146.7; 133.5; 131.3; 121.3; 120.1; 114.9;113.6; 72.0; 60.1; 52.4; 28.3; 19.5; 19.3.

C₁₆H₁₅Cl₂NO₂ (MW=324.31); mass spectroscopy (MH⁺) 325.

Example 6 Synthesis of N-(3,4-dichlorophenyl)alanine iso-butyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and iso-butyl pyruvate (prepared by following GeneralProcedure O above), the title compound was prepared as an oil. Thereaction was monitored by tlc on silica gel (Rf=0.55 in 25%EtOAc/hexanes) and purification was by preparative plate chromatography(silica gel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

1H-nmr (CDCl₃): δ=7.18 (d, 1H, J=8.7 Hz), 6.66 (d, 1H, J=2.7 Hz), 6.43(dd, 1H, J=8.7 Hz, J=2.7 Hz), 4.30 (s, 1H), 4.08 (q, 1H, J=6.9 Hz), 1.94(sept, 1H, J=6.7 Hz), 1.47 (d, 3H, J=6.9 Hz), 0.91 (d, 6H, J=6.6 Hz).

¹³C-nmr (CDCl₃) δ=174.5, 146.7, 133.5, 131.3, 121.3, 114.9, 113.6, 72.0,52.4, 28.3, 19.5, 19.3.

C₁₃H₁₇Cl₂NO₂ (MW=290.19); mass spectroscopy (MH⁺) 290.

Example 7 Synthesis of N-(3,4-dichlorophenyl)alanine iso-propyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and isopropyl pyruvate (prepared by following GeneralProcedure O above using isopropanol in place of iso-butanol), the titlecompound was prepared as an oil. The reaction was monitored by tlc onsilica gel (Rf=0.4 in 25% EtOAc/hexanes) and purification was bypreparative plate chromatography (silica gel using 25% EtOAc/hexanes asthe eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.18 (d, 1H); 6.66 (d, 1H,); 6.43 (dd, 1H); 4.30 (bs,1H); 4.08 (m, 1H); 1.94 (m, 1H); 1.47 (d, 3H); 0.91 (d, 6H).

¹³C-nmr (CDCl₃): δ=174.5; 146.7; 133.5; 131.3; 121.3; 114.9; 113.6;72.0; 52.4; 19.5.

C₁₂H15Cl₂NO₂ (MW=276.16); mass spectroscopy (MH⁺) 277.

Example 8 Synthesis of N-(3,4-dichlorophenyl)alanine n-butyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and n-butyl pyruvate (prepared by following General ProcedureO above using n-butanol in place of iso-butanol), the title compound wasprepared. The reaction was monitored by tlc on silica gel (Rf=0.7 in 25%EtOAc/hexanes) and purification was by preparative plate chromatography(silica gel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.18 (d, 1H); 6.66 (d, 1H,); 6.43 (dd, 1H); 4.30 (bs,1H); 4.2 (m, 2H); 4.08 (q, 1H); 1.94 (m, 1H); 1.47 (m, 4H); 0.91 (t,3H).

¹³C-nmr (CDCl₃): δ=174.5; 146.7; 133.5; 131.3; 121.3; 114.9; 113.6;72.0; 52.4; 28.3; 20.2; 19.5.

C₁₃H₁₇Cl₂NO₂ (MW=290.19); mass spectroscopy (MH⁺) 291.

Example 9 Synthesis of N-(3,4-dichlorophenyl)alanine methyl ester (R,Sisomers)

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and methyl pyruvate (Aldrich), the title compound was preparedas an oil. The reaction was monitored by tlc on silica gel (Rf=0.55 in25% EtOAc/hexanes) and purification was by flash chromatography (silicagel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.19 (d, J=8.73 Hz, 1H), 6.66 (d, J=2.75 Hz, 1H), 6.43(dd, J=8.73 Hz, 2.80 Hz, 1H), 4.25 (bd, J=8.25 Hz, 1H), 4.08 (m, 1H),3.76 (s, 3H), 1.47 (d, J=6.90 Hz).

¹³C-nmr (CDCl₃) δ=174.35, 145.96, 132.87, 130.70, 120.76, 114.38,112.90, 52.43, 51.70, 18.67.

C₁₀H₁₁Cl₂NO₂ (MW=248.11); mass spectroscopy (MH⁺) 247.

Example 10 Synthesis of N-(3,4dichlorophenyl)alanine cyclopentyl ester

Following transesterification General Procedure B above and usingN-(3,4-dichlorophenyl)alanine methyl ester (from Example 9 above) andcyclopentanol (Aldrich), the title compound was prepared as an oil. Thereaction was monitored by silica gel tlc (Rf=0.66 in 25% EtOAc/hexanes).Purification was by preparative plate chromatography (silica gel using25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.19 (d, 1H, J=8.7 Hz), 6.66 (d, 1H, J=2.7 Hz), 6.43(dd, 1H, J=8.7 Hz, J=2.7 Hz), 5.22 (m, 1H), 4.27 (d, 1H, J=8.1 Hz), 4.02(quint, 1H, J=7.5 Hz), 1.74 (m, 8H), 1.43 (d, 3H, J=6.9 Hz).

¹³C-nmr (CDCl₃): δ=174.3, 146.7, 133.4, 131.2, 121.2, 114.9, 113.7,78.9, 52.5, 33.2, 24.2, 24.1, 19.1.

C₁₄H₁₇Cl₂NO₂ (MW=302.20); mass spectroscopy (MH⁺) 301.

Example 11 Synthesis of N-(3,4-dichlorophenyl)alanine n-propyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and n-propyl pyruvate (prepared by following General ProcedureO above using n-propanol in place of iso-butanol), the title compoundwas prepared as an oil. The reaction was monitored by tlc on silica gel(Rf=0.5 in 25% EtOAc/hexanes) and purification was by preparative platechromatography (silica gel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.2 (d, 1H); 6.6 (d, 1H); 6.4 (dd, 1H); 4.30 (bs, 1H);4.2 (q, 2H); 4.08 (q, 1H); 1.94 (m, 2H); 1.5 (d, 3H); 0.95 (t, 3H).

¹³C-nmr (CDCl₃): δ=178; 144.7; 130.2; 120.62; 115.11; 71.82; 52.90.

C₁₂H₁₅Cl₂NO₂ (MW=276.16); mass spectroscopy (MH⁺) 277.

Example 12 Synthesis of N-(3,4-dichlorophenyl)alanine allyl ester

Following transesterification General Procedure B above and usingN-(3,4-dichlorophenyl)alanine methyl ester (from Example 9 above) andallyl alcohol (Aldrich), the title compound was prepared as an oil. Thereaction was monitored by silica gel tlc (Rf=0.62 in 25% EtOAc/hexanes).Purification was by preparative plate chromatography (silica gel using25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.19 (d, 1H, J=8.7 Hz), 6.67 (d, 1H, J=2.8 Hz), 6.44(dd, 1H, J=8.7 Hz, J=2.8 Hz), 5.90 (m, 1H), 5.30 (m, 2H), 4.64 (m, 2H),4.26 (m, 1H, 4.10 (m, 1H), 1.48 (d, 3H, J=6.9 Hz).

¹³C-nmr (CDCl₃): δ=174.1, 146.6, 133.5, 132.1, 131.3, 121.4, 119.6,115.0, 113.6, 66.5, 52.4, 19.3.

C₁₂H₁₃Cl₂NO₂ (MW=274.15); mass spectroscopy (MH⁺) 273.

Example 13 Synthesis of N-(3,4-dichlorophenyl)alanine 4-methylpentylester

Following transesterification General Procedure B above and usingN-(3,4-dichlorophenyl)alanine methyl ester (from Example 9 above) and4-methylpentanol (Aldrich), the title compound was prepared as an oil.The reaction was monitored by silica gel tlc (Rf=0.70 in 25%EtOAc/hexanes). Purification was by preparative plate chromatography(silica gel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.18 (d, 1H, δ=8.7 Hz), 6.66 (d, 1H, J=2.7 Hz), 6.43(dd, 1H, J=8.7 Hz, J=2.7 Hz), 4.28 (m, 1H), 4.10 (m, 3H), 1.55 (m, 6H),1.19 (m, 2H), 0.87 (d, 3H, J=6.6 Hz).

¹³C-nmr (CDCl₃): δ=174.6, 146.7, 133.4, 131.3, 121.3, 115.0, 113.6,66.4, 52.4, 35.4, 28.2, 27.0, 23.0, 19.3.

C₁₅H₂₁Cl₂NO₂ (MW=318.25); mass spectroscopy (MH⁺) 317.

Example 14 Synthesis of N-(3,4-dichlorophenyl)alanine2,2-dimethyl-1,3-dioxolane-4-methyl ester

Following transesterification General Procedure B above and usingN-(3,4-dichlorophenyl)alanine methyl ester (from Example 9 above) and2,2-dimethyl-1,3-dioxolane-4-methanol (solketal) (Aldrich), the titlecompound was prepared as a mixture of diastereomers. The reaction wasmonitored by silica gel tdc (Rf=0.32 in 25% EtOAc/hexanes). Purificationwas by preparative plate chromatography (silica gel using 25%EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.19 (d, 1H, J=8.7 Hz), 6.66 (d, 1H, 2.7 Hz), 6.43(dd, 1H, J=8.7 Hz, J=2.7 Hz), 4.22 (m, 6H), 3.70 (m, 1H), 1.43 (m, 9H).

¹³C-nmr (CDCl₃): δ=174.34, 174.32, 146.5, 133.5, 131.3, 121.5, 115.0,113.6, 110.52, 110.51, 73.97, 73.89, 66.6, 66.01, 65.95, 52.42, 52.37,27.3, 25.8, 19.3.

C₁₅H₁₉Cl₂NO₄ (MW=348.23); mass spectroscopy (MH⁺) 347.

Example 15 Synthesis of N-(3,4-dichlorophenyl)alanine cyclohexylmethylester

Following transesterification General Procedure B above and usingN-(3,4-dichlorophenyl)alanine methyl ester (from Example 9 above) andcyclohexylmethanol (Aldrich), the title compound was prepared.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.19 (d, 1H), 6.68 (d, 1H), 6.45 (dd, 1H), 4.26 (bd,1H), 4.10 (m, 1H), 3.95 (d, 2H), 1.70-1.55 (m, 6H), 1.50 (d, 3H),1.35-0.85 (m, 5H).

¹³C-nmr (CDCl₃): δ=174.58, 146.72, 133.48, 131.27, 121.34, 114.98,113.72, 71.06, 52.52, 37.68, 30.10, 26.83, 26.17, 19.32.

C₁₅H₂₁Cl₂NO₂ (MW=318.25); mass spectroscopy (MH⁺) 317.

Example 16 Synthesis of N-(3,4-dichlorophenyl)alaninetert-butyloxycarbonylmethyl ester

Following General Procedure E above and usingN-(3,4-dichlorophenyl)alanine (from Example B above) and tert-butylbromoacetate (Aldrich), the title compound was prepared as a solid. Thereaction was monitored by silica gel tlc (Rf=0.57 in 25% EtOAc/hexanes).Purification was by recrystallization from ethanol.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.19 (d, 1H), 6.68 (d, 1H), 6.45 (dd, 1H), 4.55 (m,2H), 4.20 (m, 2H), 1.55 (d, 3H), 1.45 (s, 9H).

¹³C-nmr (CDCl₃): δ=173.9, 166.9, 146.5, 133.5, 131.3, 115.1, 113.6,83.4, 62.2, 52.2, 28.6, 19.3.

C₁₅H₁₉Cl₂NO₄ (MW=348.23); mass spectroscopy (MH⁺) 347.

Example 17 Synthesis of N-(3,4-dichlorophenyl)leucine iso-butyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and iso-butyl 4-methyl-2-oxopentanoate (prepared by followingGeneral Procedure O above using 4-methyl-2-oxovaleric acid (Fluka) andiso-butanol), the title compound was prepared as an oil. The reactionwas monitored by tlc on silica gel (Rf=0.6 in 25% EtOAc/hexanes) andpurification was by preparative plate chromatography (silica gel using25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.2 (d, 1H); 6.5 (d, 1H); 6.4 (dd, 1H); 4.30 (bs, 1H);4.08 (q, 1H); 3.8(m, 2H); 1.8 (m, 3H); 0.91 (m, 12H).

¹³C-nmr (CDCl₃): δ=174.5; 146.7; 133.5; 131.3; 121.3; 114.9; 113.6;72.0; 52; 28.3; 20.1; 19.5.

C₁₆H₂₃Cl₂NO₂ (MW=332.27); mass spectroscopy (MH⁺) 333.

Example 18 Synthesis of 2-[N-(3,4-dichlorophenyl)amino]pentanoic acidiso-butyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and iso-butyl 2-oxopentanoate (prepared by following GeneralProcedure O above using 2-oxovaleric acid (Fluka) and iso-butanol), thetitle compound was prepared as an oil. The reaction was monitored by tlcon silica gel (Rf=0.5 in 25% EtOAc/hexanes) and purification was bypreparative plate chromatography (silica gel using 25% EtOAc/hexanes asthe eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.2 (d, 1H); 6.6 (d, 1H); 6.4 (dd, 1H); 4.3 (d, 1H);3.8 (m, 3H); 1.9 (m, 6H); 1.0 (t, 3H), 0.9 (m, 6H).

¹³C-nmr (CDCl₃): δ=178; 144.7; 130.2; 120.62; 115.11; 71.82; 52.90;28.30; 19.53.

C₁₅H₂₁Cl₂NO₂ (MW=318.3); mass spectroscopy (MH⁺) 319.

Example 19 Synthesis of N-(4-cyanophenyl)alanine iso-butyl ester

Following General Procedure P above and using 4-fluorobenzonitrile(Aldrich) and D,L-alanine iso-butyl ester hydrochloride (from Example Aabove), the title compound was prepared as an oil. The product wasrecovered by column chromatography on silica gel using 1:5 EtOAc/hexanesas the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.44 (d, J=8.8 Hz, 2H), 6.57 (d, J=8.8 Hz, 2H), 4.74(d, J=8.1 Hz, 1H), 4.18 (t, J=7.4 Hz, 1H), 3.95 (m, 2H), 1.94 (m, 1H),1.51 (d, δ=6.9 Hz, 3H), 0.91 (d, J=6.7 Hz, 6H).

¹³C-nmr (CDCl₃): δ=173.4, 149.7, 133.8, 120.1, 112.7, 99.8, 71.6, 51.2,27.7, 18.9, 18.6.

C₁₄H₁₈N₂O₂ MW=246.31; mass spectroscopy (MH⁺) 247.

Example 20 Synthesis of N-(3-chloro-4-cyanophenyl)alanine iso-butylester

Following General Procedure P above and using2-chloro-4-fluorobenzonitrile (Aldrich) and D,L-alanine iso-butyl esterhydrochloride (from Example A above), the title compound was prepared.The product was recovered by column chromatography on silica gel using1:5 EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.40 (d, J=8.5 Hz, 1H), 6.62 (d, J=2.3 Hz, 1H), 6.48(dd, J=2.4, 8.6 Hz, 1H), 4.90 (d, J=7.6 Hz, 1H), 4.16 (quintet, J=7.1Hz, 1H), 3.96 (dd, J=2.2, 6.7 Hz, 2H), 1.97 (m, 1H), 1.51 (d, J=7.0 Hz,3H), 0.93 (d, J=6.7 Hz, 6H).

¹³C-nmr (CDCl₃): δ=173.0, 150.4, 138.3, 134.9, 117.3, 112.8, 111.3,100.6, 71.7, 51.1, 27.7, 18.9, 18.4.

C₁₄H₁₇N₂O₂Cl MW=280.76; mass spectroscopy (MH⁺) 281.

Example 21 Synthesis of N-(3,4-dichloro)alanine iso-butyl ester (Sisomer)

Following General Procedure M above and using 3,4-dichloroaniline(Aldrich) and iso-butyl R-(+)-lactate (Aldrich), the title compound wasprepared as an oil. The reaction was monitored by silica gel tlc(Rf=0.55 in 25% EtOAc/hexanes). Purification was column chromatography.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.19 (d, J=8.73, 1H), 6.67 (d, J=2.75, 1H), 6.45 (dd,J=8.73, J=2.75, 1H), 4.28 (bd, J=8.36, 1H), 4.09 (quint, 1H), 3.94 (d,J=6.66, 2H), 1.95 (hept, J=6.71, 1H), 1.49 (d, J=6.90, 3H), 0.92 (d,J=6.04, 6H).

¹³C-nmr (CDCl₃): δ=174.57, 146.67, 133.47, 131.28, 121.29, 114.93,113.63, 71.01, 52.43, 28.30, 19.55, 19.33.

C₁₃H₁₇Cl₂NO₂ (MW=290.19); mass spectroscopy (MH⁺) 290.

Example 22 Synthesis of N-(3,4-dichloro)alaninetetrahydrofuran-3-yl-methyl ester

Following transesterification General Procedure B above and usingN-(3,4-dichlorophenyl)alanine methyl ester (from Example 9 above) andtetrahydro-3-furanmethanol (Aldrich), the title compound was prepared asan oil. The reaction was monitored by silica gel tlc (Rf=0.33 in 25%EtOAc/hexanes). Purification was by preparative plate chromatography(silica gel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ7.18 (d, 1H, J=8.7 Hz), 6.65 (d, 1H, J=2.7 Hz), 6.42(dd, 1H, J=8.7 Hz, J=2.7 Hz), 4.30 (m, 1H), 4.09 (m, 3H), 3.78 (m, 3H),3.53 (m, 1H), 2.56 (m, 1H), 1.94 (m, 1H), 1.58 (m, 1H), 1.46 (d, 3H,J=6.9 Hz).

¹³C-nmr (CDCl₃): δ=174.5, 146.6, 133.5, 131.3, 121.4, 114.9, 113.6,70.86, 70.83, 68.2, 67.31, 67.29, 52.4, 38.7, 29.36, 29.33, 19.2.

C₁₄H₁₇Cl₂NO₃ (MW=318.20); mass spectroscopy (MH⁺) 318.

Example 23 Synthesis of N-(3,5-dichlorophenyl)alanine n-propyl ester

Following General Procedure A above and using 3,5-dichloroaniline(Aldrich) and n-propyl pyruvate (which can be prepared by followingGeneral Procedure O above using n-propanol in place of iso-butanol), thetitle compound could be prepared.

Example 24 Synthesis of 2-[N-(3,4-dichlorophenyl)amino]butanoic acidiso-butyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and iso-butyl 2-oxobutanoate (prepared by following GeneralProcedure O above using 2-oxobutyric acid (Aldrich) and iso-butanol),the title compound was prepared as an oil. The reaction was monitored bytlc on silica gel (Rf=0.3 in 25% EtOAc/hexanes) and purification was bypreparative plate chromatography (silica gel using 25% EtOAc/hexanes asthe eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.2 (d, 1H); 6.6 (d, 1H); 6.4 (dd, 1H); 4.3 (d, 1H);3.8 (m, 3H); 1.9 (m, 3H); 1.0 (t, 3H); 0.9(m, 6H).

¹³C-nmr (CDCl₃): δ=178; 144.7; 130.2; 120.62; 115.11; 71.82; 52.90;28.30; 20.5; 19.53.

C₁₄H₁₉Cl₂NO₂ (MW=304.22); mass spectroscopy (MH⁺) 305.

Example 25 Synthesis of N-(4-chlorophenyl)alanine iso-butyl ester

Following General Procedure A above and using 4-chloroaniline (Aldrich)and iso-butyl pyruvate (prepared by following General Procedure Oabove), the title compound was prepared as an oil. The reaction wasmonitored by tlc on silica gel (Rf=0.6 in 25% EtOAc/hexanes) andpurification was by preparative plate chromatography (silica gel using25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.18 (d, 2H), 6.66 (d, 2H), 4.30 (bs, 1H), 4.08 (q,1H), 1.94 (sept, 1H), 1.47 (d, 3H), 0.91 (d, 6H).

¹³C-nmr (CDCl₃): δ=174.5, 146.7, 133.5, 131.3, 121.3, 114.9, 113.6,72.0, 52.4, 28.3, 19.5, 19.3.

C₁₃H₁₈ClNO₂ (MW=255.75); mass spectroscopy (MH⁺) 256.

Example 26 Synthesis of N-(3,5-dichlorophenyl)alanine isobutyl ester

Following General Procedure A above and using 3,5-dichloroaniline(Aldrich) and iso-butyl pyruvate (prepared by following GeneralProcedure O above), the title compound was prepared as an oil. Thereaction was monitored by tlc on silica gel (Rf=0.4 in 25%EtOAc/hexanes) and purification was by preparative plate chromatography(silica gel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.18 (d, 2H), 6.66 (m, 1H), 4.30 (bs, 1H), 4.08 (q,1H), 1.94 (m, 1H), 1.47 (d, 3H), 0.91 (d, 6H).

¹³C-nmr (CDCl₃): δ=175; 146.7; 133; 131; 121; 114.9; 112.6; 72.0; 52.4;28.3; 19.5.

C₁₃H₁₇Cl₂NO₂ (MW=290.2); mass spectroscopy (MH⁺) 291.

Example 27 Synthesis of N-(4-ethylphenyl)alanine methyl ester

A solution of 0.68 g (5 mmol) of 4′-aminoacetophenone (Aldrich), 0.60 mLof 90% methyl pyruvate (Aldrich) and 0.05 g (0.25 mmol) ofp-toluenesulfonic acid in ethanol was hydrogenated in the presence of acatalytic amount of 10% Pd/C at from 30 to 15 psi of hydrogen for 16hours. The catalyst was removed by filtering the reaction mixturethrough Celite and the solvent was evaporated to provide the crudeproduct. The product was purified by column chromatography (silica gelusing 1:9 EtOAc/hexanes as the eluant) to provide the title compound.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=1.19 (t, J=7.6 Hz, 3H), 1.47 (d, J=6.8 Hz, 3H), 2.54(q, J=7.6 Hz, 2H), 3.74 (s, 3H), 4.04 (bs, 1H), 4.13 (m, 1H), 6.57 (d,J=8.5 Hz, 2H), 7.03 (d, J=8.4 Hz, 2H).

¹³C-nmr (CDCl₃): δ=15.8, 18.0, 27.9, 52.17, 52.19, 113.5, 128.6, 134.1,144.4, 175.3.

C₁₂H₁₇NO₂ MW=207.27; mass spectroscopy (MH⁺) 208.

Example 28 Synthesis of N-(4-(1-ethoxy)ethylphenyl)alanine methyl ester

Following the procedure for Example 27 above, the title compound wasisolated as another reaction product by column chromatography (silicagel using 1:9 EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=1.15 (t, J=7.0 Hz, 3H), 1.40 (d, J=6.5 Hz, 3H), 1.47(d, J=6.1 Hz, 3H), 3.31 (q, J=5.1 Hz, 2H), 3.74 (s, 3H), 4.14 (m, 2H),4.29 (q, J=6.4 Hz, 1H), 6.57 (d, J=8.5 Hz, 2H), 7.12 (d, J=8.4 Hz, 2H).

¹³C-nmr (CDCl₃): δ=15.4, 19.0, 23.9, 51.9, 52.2, 63.4, 77.3, 113.1,127.3, 133.6, 145.8, 175.1.

C₁₄H₂₁NO₃ MW=251.33; mass spectroscopy (MH⁺) 251.

Example 29 Synthesis of N-(3,4-dichloro)alanine 2,2-dimethylpropyl ester(R,S isomers)

Following transesterification General Procedure Q above and usingN-(3,4-dichlorophenyl)alanine methyl ester (from Example 9 above) andneopentyl alcohol (Aldrich), the title compound was prepared. Thereaction was monitored by silica gel tlc (Rf=0.72 in 25% EtOAc/hexanes).Purification was by flash chromatography (silica gel using 25%EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.19 (d, 1H, J=8.7 Hz), 6.68 (d, 1H, J=2.7 Hz), 6.45(dd, 1H, J=8.7 Hz, J=2.7 Hz), 4.29 (m, 1H), 4.11 (m, 1H), 3.85 (m, 2H),1.49 (d, 3H, J=6.9 Hz), 0.93 (s, 9H).

¹³C-nmr (CDCl₃): δ=174.6, 146.7, 133.5, 131.3, 121.3, 114.9, 113.7,75.2, 52.4, 32.0, 26.9, 19.4.

C₁₄H₁₉Cl₂NO₂ (MW=304.22); mass spectroscopy (MH⁺) 303.

Example 30 Synthesis of N-(3,4-dichlorophenyl)glycine iso-butyl ester

3,4-Dichloroaniline (Aldrich) was treated with di-tert-butyl dicarbonate(Aldrich) using conventional procedures to produce the N-BOC aniline.The N-BOC aniline was treated with sodium hydride in THF and then withisobutyl 2-bromoacetate (from Example D above) to produce the N-BOCN-(3,4-dichlorophenyl)glycine iso-butyl ester. The BOC group was thenremoved using General Procedure N above to afford the title compound.The reaction was monitored by tlc on silica gel (Rf=0.78 in 50%EtOAc/hexanes) and purification was by preparative plate chromatography(silica gel using 50% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.19 (dd, J=4.1, 4.7, 3.4, 1H); 6.65 (d, J=2.7, 1H);6.44 (dd, J=2.7, 4.5, 4.2, 1H): 4.4 (m, 1H): 3.97 (dd, J=3.6, 3.0, 2.3,2H); 3.87 (s, 2H); 1.9 (m, 1H); 0.93 (d, J=6.7, 6H).

¹³C-nmr (CDCl₃): δ=171.2, 147.0, 133.5, 131.3, 121.2, 114.5, 113.3,72.2, 46.0, 28.2, 19.6.

C₁₂H₁₅Cl₂NO₂ (MW=276); mass spectroscopy (MH⁺) 277.

Example 31 Synthesis of N-(3,4-dichlorophenyl)alanine 2-ethylbutyl ester

Following General Procedure A above and using 3,4-dichloroaniline(Aldrich) and 2-ethylbutyl pyruvate (prepared by following GeneralProcedure O above using 2-ethylbutanol (Aldrich) in place ofiso-butanol), the title compound was prepared as an oil. The reactionwas monitored by tlc on silica gel (Rf=0.6 in 25% EtOAc/hexanes) andpurification was by preparative plate chromatography (silica gel using25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.2 (d, 1H); 6.6 (d, 1H); 6.4 (dd, 1H); 4.2 (t, 2H);4.1 (q, 1H); 1.5 (d, 3H); 1.4 (m, 4H); 1.0 (m, 6H).

¹³C-nmr (CDCl₃): δ=178; 144.7; 130.2; 120.62; 115.11; 70.7; 51.90; 26.3;19.53, 18.5.

C₁₅H₂₁Cl₂NO₂ (MW=318.25); mass spectroscopy (MH⁺) 319.

Example 32 Synthesis of N-(3-chloro-4-iodophenyl)alanine iso-butyl ester

Following General Procedure R above and using 3-chloro-4-iodoaniline(Aldrich), N-BOC-3-chloro-4-iodoaniline was prepared. To a stirredslurry of 5.0 equivalents of sodium hydride in DMF was added 1.0equivalent of N-BOC-3-chloro-4-iodoaniline and then 1.1 equivalents ofiso-butyl 2-bromopropionate (from Example D above) were slowly added.The reaction was heated to 100° C. for 10 hours, cooled, diluted withdichloromethane and washed with cold 1N HCl, water and brine. Thesolvents were removed at reduced pressure and the residue waschromatographed to provide N-BOC-N-(3-chloro-4-iodophenyl)alanineiso-butyl ester as a clear oil. Following General Procedure N above, theBOC group was removed from N-BOC-N-(3-chloro-4-iodophenyl)alanineiso-butyl ester to provide the title compound. The BOC-removal reactionwas monitored by tlc on silica gel (Rf=0.58 in 30% EtOAc/hexanes) andpurification was by preparative plate chromatography (silica gel using30% EtOAc/hexanes as the eluant). The compound was further purified bychromatography on an HPLC chiral column (Chiralcel OD).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.52 (d, J=8.7, 1H); 6.72 (d, J=2.7, 1H); 6.25 (dd,J=2.7, 5.9, 2.7, 1H); 4.35 (d, J=6.6, 1H): 4.08 (quintex, J=7.2, 6.7,1H); 3.93 (d, J=6.7, 2H): 1.94 (m, 1H); 1.47 (d, J=6.9, 3H); 0.92 (d,J=6.9, 6H).

¹³C-nmr (CDCl₃): δ=174.5, 148.3, 140.7, 139.5, 114.4, 114.3, 82.6, 72.0,52.2, 28.3, 19.6, 19.3.

C₁₃H₁₇ClINO₂ (MW=381.5); mass spectroscopy (MH⁺) 382.

Example 33 Synthesis of N-(4-azidophenyl)alanine iso-butyl ester

Following General Procedure A above and using 4-azidoaniline (Aldrich)and iso-butyl pyruvate (prepared by following General Procedure Oabove), the title compound was prepared as an oil. The reaction wasmonitored by tlc on silica gel (Rf=0.3 in 25% EtOAc/hexanes) andpurification was by preparative plate chromatography (silica gel using25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.3 (d, 2H), 6.8 (d, 2H), 4.30 (bs, 1H), 4.08 (q, 1H),1.94 (sept, 1H), 1.47 (d, 3H), 0.91 (d, 6H).

¹³C-nmr (CDCl₃): δ=174.5, 148.7, 131.5, 130.3, 121.3, 114.9, 113.6,72.0, 52.4, 28.3, 19.5, 19.3.

C₁₃H₁₈N₄O₂ (MW=262.31); mass spectroscopy (MH⁺) 263.

Example 34 Synthesis of N-[(4-phenylcarbonyl)phenyl]alanine iso-butylester

Following General Procedure A above and using 4′-aminobenzophenone(Aldrich) and iso-butyl pyruvate (prepared by following GeneralProcedure O above), the title compound was prepared as an oil. Thereaction was monitored by tlc on silica gel (Rf=0.4 in 25%EtOAc/hexanes) and purification was by preparative plate chromatography(silica gel using 25% EtOAc/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.7 (d, 2H), 7.1 (m, 5H), 6.9 (d, 2H), 4.30 (bs, 1H),4.08 (q, 1H), 1.94 (sept, 1H), 1.47 (d, 3H), 0.91 (d, 6H).

¹³C-nmr (CDCl₃): δ=199, 178.5, 149.7, 131.5, 130.3, 126, 121.3, 114.9,113.6, 72.0, 52.4, 28.3, 19.5, 19.3.

C₂₀H₂₃NO₃ (MW=325.41); mass spectroscopy (MH⁺) 326.

Example 35 Synthesis of N-(3,5-difluorophenyl)alanine iso-butyl ester

Following General Procedure H above and usingN-(3,5-difluorophenyl)alanine (from Example C above) and isobutanol, thetitle compound was prepared as an oil. The reaction was monitored by tlcon silica gel (Rf=0.9 in 3% methanol/methylene chloride) andpurification was by preparative plate chromatography (silica gel using3% methanol/methylene chloride as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=6.1 (m, 3H), 4.5 (bs, 1H), 4.1 (d, 1H), 3.95 (m, 2H),2.0 (m, 1H), 1.5 (d, J=7 Hz, 3H), 0.95(d, J=6 Hz, 6H).

¹³C-nmr (CDCl₃): δ=174.44, 166.40, 166.19, 163.16, 162.95, 149.43,96.73, 96.60, 96.48, 96.35, 94.06, 93.72, 93.37, 72.03, 52.30, 28.29,19.47, 19.23.

C₁₃H₁₇F₂NO₂ (MW=290.2); mass spectroscopy (MH⁺) 291.

Example 36 Synthesis of N-(3,4-dichlorophenyl)alanineO-acylacetamidoxime ester

Following General Procedure K above and usingN-(3,4-dichlorophenyl)alanine (from Example B above) and acetamide oxime(prepared according to the procedures described in J. Org. Chem., 46,3953 (1981)), the title compound was prepared as a semisolid. Thereaction was monitored by tlc on silica gel (Rf=0.4 in ethyl acetate)and purification was by preparative plate chromatography (silica gelusing ethyl acetate as the eluant).

NMR data was as follows:

¹H-nmr (DMSO-d₆): δ=7.27 (d, 1H), 6.81 (s, 1H) 6.4 (broad s, 2H), 6.62(d, 1H), 6.45 (d, 1H), 4.22 (m, 1H), 1.74 (s, 3H), 1.40 (d, 3H).

C₁₁H₁₃Cl₂N₃O₂ (MW=290.15); mass spectroscopy (MH⁺) 291.

Example 37 Synthesis of N-(3,4-dichlorophenyl)alanine pyrrolyl amide

Following General Procedure L above and usingN-(3,4-dichlorophenyl)alanine (from Example B above) and pyrrole(Aldrich), the title compound was prepared as an oil. The reaction wasmonitored by tlc on silica gel (Rf=0.28 in 10% ethyl acetate/hexanes)and purification was by preparative plate chromatography (silica gelusing 10% ethyl acetate/hexanes as the eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.36 (d, J=2.2, 2H); 7.20 (d, J=8.7, 1H); 6.71 (d,J=2.7, 1H); 6.5 (m, 1H); 6.38 (t, J=2.4, 2H); 4.8 (m, 1H); 4.57 (d,J=8.7, 1H); 1.59 (d, J=6.8, 3H).

¹³C-nmr (CDCl₃): δ=171.9, 146.1, 133.6, 131.5, 121.9, 119.6, 115.4,114.7, 113.8, 51.8, 20.2.

C₁₃H₁₂Cl₂N₂O (MW=283); mass spectroscopy (MH⁺) 284.

Example 38 Synthesis of N-(3,4-dichlorophenyl)alanineO-acylbutyramideoxime ester

Following General Procedure I above and usingN-(3,4-dichlorophenyl)alanine 2,4,6-trichlorophenyl ester (prepared fromN-(3,4-dichlorophenyl)alanine methyl ester (from Example 9) usingessentially the same procedure as described in Example E above) andbutyramide oxime (prepared according to the procedures described in J.Org. Chem., 46, 3953 (1981)), the title compound was prepared as asemisolid. The reaction was monitored by tlc on silica gel (Rf=0.25 in50% ethyl acetate/hexanes) and purification was by preparative platechromatography (silica gel using 50% ethyl acetate/hexanes as theeluant).

NMR data was as follows:

¹H-nmr (d₆-DMSO): δ=7.27 (d, 1H), 6.83 (s, 1H) 6.38 (broad s, 2H), 6.61(d, 1H), 6.46 (d, 1H), 4.25 (m, 1H), 2.02 (t, 2H), 1.55 (m, 2H), 1.40(d, 3H), 0.88 (t, 3H).

C₁₃H₁₇Cl₂N₃O₂ (MW=318.20); mass spectroscopy (MH⁺) 319.

Example 39 Synthesis of 2-[N-(naphth-2-yl)amino]butanoic acid ethylester

Following General Procedure J above and using 2-aminonaphthalene(Aldrich) and ethyl 2-bromobutyrate (Aldrich), the title compound wasprepared as a solid, m.p. 81-83° C. The reaction was monitored by silicagel tlc (Rf=0.5 in CHCl₃). Purification was by chromatography (silicagel using chloroform as the eluant).

NMR data was as follows: ¹H-nmr (d⁶-DMSO): δ=7.63 (m, 2H), 7.54 (d, 1H),7.31(t, 1H), 7.12 (t, 1H), 7.03 (d, 1H), 6.62 (s, 1H), 6.32 (d, 1H),4.15 (m, 3H), 1.42 (d, 3H), 1.19 (t, 3H).

C₁₆H₁₉NO₂ (MW=257.34); mass spectroscopy (MH⁺) 258.

Example 40 Synthesis of N-(2-naphthyl)alanine iso-butyl ester

Following General Procedure A above and using 2-aminonaphthalene(Aldrich) and isobutyl pyruvate (prepared by following General ProcedureO above), the title compound was prepared as an oil. Purification was bypreparative plate chromatography (silica gel using 25% EtOAc/hexanes asthe eluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.65 (m, 3H), 7.38 (t, 1H, J=6.9 Hz), 7.23 (t, 1H,J=6.9 Hz), 6.93 (m, 1H), 6.81 (d, 1H, J=2.3 Hz), 4.31 (q, 1H, J=6.9 Hz),3.95 J=6.7 Hz, J=1.6 Hz), 1.96 (sept, 1H, J=6.7 Hz), 1.57 (d, 3H, J=6.9Hz), 0.93 (dd, 6H, J=6.7 Hz, J=1.6 Hz).

¹³C-nmr (CDCl₃) δ=174.6, 144.2, 134.9, 129.1, 127.8, 127.6, 126.3,126.0, 122.3, 118.1, 105.3, 71.2, 52.0, 27.7, 18.9, 18.8.

Example 41 Synthesis of N-(2-methylquinolin-6-yl)alanine iso-butyl ester

Following General Procedure A above and using 6-amino-2-methylquinoline(Lancaster) and iso-butyl pyruvate (prepared by following GeneralProcedure O above), the title compound was prepared. The reaction wasmonitored by silica gel tlc (Rf=0.44 in 50% EtOAc/hexanes). Purificationwas by flash chromatography (silica gel using 50% EtOAc/hexanes as theeluant).

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.90 (m, 2H), 7.10 (m, 2H), 6.66 (d, 1H, J=2.6), 4.50(bd, 1H), 4.24 (m, 1H), 3.91 (d, 2H, J=6.6 Hz), 2.64 (s, 3H), 1.91(sept, 1H, J=6.7 Hz), 1.52 (d, 3H, J=6.9 Hz), 0.87 (d, 6H, J=6.7 Hz).

¹³C-nmr (CDCl₃) δ=175.0, 155.4, 144.6, 143.4, 134.9, 130.2, 128.4,122.8, 121.8, 104.9, 71.8, 52.7, 28.3, 25.4, 19.5, 19.4.

C₁₇H₂₂Cl₂N₂O₂ (MW=286.38); mass spectroscopy (MH⁺) 287.

Example 42 Synthesis of N-(3,4-methylenedioxyphenyl)alanine iso-butylester

Following reductive amination General Procedure A above and using3,4-methylenedioxyaniline (Aldrich) and methyl pyruvate (Aldrich),N-(3,4-methylenedioxyphenyl)alanine methyl ester was prepared. Themethyl ester was then transesterified following General Procedure Qabove and using iso-butanol to provide the title compound as an oil. Thereaction was monitored by silica gel tlc (Rf=0.61 in 25% EtOAc/hexanes).Purification was by preparative plate chromatography with silica gelusing 25% EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=6.63 (d, 1H, 8.3 Hz), 6.25 (d, 1H, J=2.3 Hz), 6.04(dd, 1H, J=8.3 Hz, J=2.3 Hz), 5.83 (s, 2H), 3.96 (m, 4H), 1.92 (sept,1H, J=6.7 Hz), 1.44 (d, 3H, J=6.9 Hz), 0.90 (d, 6H, J=6.6 Hz).

¹³C-nmr (CDCl₃): δ=175.4, 148.9, 142.9, 140.8, 109.2, 105.8, 101.2,97.4, 71.6, 53.6, 28.3, 19.6, 19.5.

C₁₄H₁₉NO₄ (MW=265.31); mass spectroscopy (MH⁺) 265.

Example 43 Synthesis of N-(3,4-ethylenedioxyphenyl)alanine iso-butylester

Following reductive amination General Procedure A above and using1,4-benzodioxa-6-amine (Aldrich) and methyl pyruvate (Aldrich),N-(3,4-ethylenedioxyphenyl)alanine methyl ester was prepared. The methylester was then transesterified following General Procedure Q above usingisobutanol to provide the title compound. Purification was bypreparative plate chromatography.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=0.91 (d, J=7 Hz, 6H), 1.42 (d, J=7 Hz, 3H), 1.8-2.0(m, 1H), 3.8-3.95 (m, 3H), 4.0-4.1 (m, 1H), 4.15-4.25 (m, 4H), 6.12-6.2(m, 2H), 6.65-6.75 (m, 1H).

¹³C-nmr (CDCl₃): δ=19.55, 19.56, 19.67, 28.3, 53.4, 64.7, 65.3, 71.7,103.1, 108.0, 118.3, 142.1, 144.6, 175.4.

C₁₅H₂₁NO₄ (MW=279.34); mass spectroscopy (MH⁺) 280.

Example 44 Synthesis of N-(2-naphthyl)alanine methyl ester

Following reductive amination General Procedure A above and using2-aminonaphthalene (Aldrich) and methyl pyruvate (Aldrich), the titlecompound was prepared. The reaction was monitored by silica gel tlc(Rf=0.50 in 25% EtOAc/hexanes). Purification was by flash chromatographywith silica gel using 25% EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.65 (m, 3H), 7.48 (m, 1H), 7.25 (m, 1H), 6.91 (m,1H), 6.79 (m, 1H), 4.31 (m, 2H), 3.76 (s, 3H), 1.55 (d, 3H).

¹³C-nmr (CDCl₃): δ=175.66, 144.78, 135.55, 129.78, 128.47, 128.22,126.96, 126.67, 123.01, 118.66, 105.88, 52.95, 52.51, 19.45.

C₁₄H₁₅NO₂ (MW=229.28); mass spectroscopy (MH⁺) 229.

Example 45 Synthesis of N-(benzothiazol-6-yl)alanine ethyl ester

To a solution of 6-aminobenzothiazole (Lancaster) in dichloromethane wasadded 1.2 equivalents of pyridine, followed by 1.5 equivalents oftrifluoroacetic anhydride. The reaction was stirred at room temperaturefor 3 hours and then washed with 5% citric acid, dried over MgSO₄, andstripped free of solvent on a rotary evaporator to yield6-trifluoroacetamidothiazole. This material was dissolved in THF andthen added to a suspension of KH in THF at 0° C. A catalytic amount of18-crown-6 was added, followed by ethyl 2-bromopropionate (Aldrich). Thereaction was held at room temperature for 1 hour, and then heated toreflux for 24 hours, and then cooled to room temperature. The reactionmixture was stripped free of solvent on a rotary evaporator and theresulting residue was dissolved in ether. This solution was washed withwater, saturated aqueous NaCl, and dried over MgSO₄. The solution wasstripped free of solvent on a rotary evaporator and the title compoundwas obtained by chromatography of the residue using 5%methanol/dichloromethane (Rf=0.59) as the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=8.69 (s, 1H), 7.90 (d, 1H, J=8.8 Hz), 7.04 (d, 1H,J=2.3 Hz), 6.84 (dd, 1H, J=8.8 Hz, J=2.4 Hz), 4.41 (bd, 1H, J=7.5 Hz),4.20 (m, 3H), 1.53 (d, 3H, J=6.9 Hz), 1.27 (t, 3H, J=7.1 Hz).

¹³C-nmr (CDCl₃): δ=174.9, 150.2, 147.1, 145.6, 136.3, 124.6, 115.7,103.5, 61.9, 52.9, 19.4, 14.8.

C₁₂H₁₄N₂O₂S (MW=250.32); mass spectroscopy (MH⁺) 251.

Example 46 Synthesis of N-(indol-5-yl)alanine iso-butyl ester (S isomer)

Following General Procedure M and using 5-aminoindole (Aldrich) andiso-butyl R-(+)-lactate (Aldrich), the title compound was prepared as anoil. The reaction was monitored by silica gel tlc (Rf=0.46 in 33%EtOAc/hexanes). Purification was by preparative plate chromatographywith silica gel using 33% EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=8.11 (bs, 1H), 7.07 (d, J=8.8 Hz, 1H), 6.98 (d, J=2.8Hz, 1H), 6.83 (d, J=2.2 Hz, 1H), 6.61 (m, 1H), 6.32 (m, 1H), 4.18 (q,J=6.9 Hz, 1H), 3.95 (bs, 1H), 3.87 (d, J=6.7 Hz, 2H), 1.89 (hept, J=6.7Hz, 1H), 1.48 (d, J=6.96 Hz, 3H), 0.86 (dd, J=6.7 Hz, J=1.6 Hz, 6H).

¹³C-nmr (CDCl₃): δ=176.15, 141.06, 131.28, 129.24, 125.34, 113.34,112.53, 104.21, 102.17, 71.65, 54.28, 28.36, 19.87, 19.62.

C₁₅H₂₀N₂O₂ (MW=260.34); mass spectroscopy (MH⁺) 261.

Example 47 Synthesis of N-(naphth-2-yl)alanine O-acylacetamidoxime ester

Following General Procedure I above using N-(naphth-2-yl)alanine2,4,6-trichlorophenyl ester (from Example E above) and acetamide oxime(prepared according to the procedures described in J. Org. Chem., 46,3953 (1981)), the title compound was prepared as a semisolid. Thereaction was monitored by tlc on silica gel (Rf=0.4 in ethyl acetate)and purification was by preparative plate chromatography (silica gelusing ethyl acetate as the eluant).

NMR data was as follows:

¹H-nmr (d⁶-DMSO): δ=7.64 (t, 2H), 7.54 (d, 1H), 7.32 (t, 1H), 7.13 (t,1H), 7.04 (d, 1H), 6.78 (s, 1H) 6.42 (broad s, 2H), 6.32 (d, 1H), 4.33(m, 1H), 1.72 (s, 3H), 1.46 (d, 3H).

C₁₅H₁₇N₃O₂ (MW=271.32); mass spectroscopy: 271.

Example 48 Synthesis of N-(2-naphthyl)alanine ethyl ester

Following reductive amination General Procedure A above and using2-aminonaphthalene (Aldrich) and ethyl pyruvate (Aldrich), the titlecompound was prepared as a solid having a melting point of 52-56° C. Thereaction was monitored by silica gel tlc (Rf=0.50 in 25% EtOAc/hexanes).Purification was by flash chromatography with silica gel using 25%EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.65 (m, 3H), 7.48 (m, 1H), 7.25 (m, 1H), 6.91 (m,1H), 6.79 (m, 1H), 4.31 (m, 2H), 3.76 (s, 3H), 1.55 (d, 3H).

¹³C-nmr (CDCl₃): δ=175.66, 144.78, 135.55, 129.78, 128.47, 128.22,126.96, 126.67, 123.01, 118.66, 105.88, 52.95, 52.51, 19.45.

C₁₄H₁₅NO₂ (MW=229.28); mass spectroscopy (MH⁺) 229.

Example 49 Synthesis of N-(3,4-dichlorophenyl)alanineO-acylpropionamidoxime ester

Following General Procedure I above using N-(3,4-dichlorophenyl)alanine2,4,6-trichlorophenyl ester (prepared from N-(3,4-dichlorophenyl)alaninemethyl ester (from Example 9) using essentially the same procedure asdescribed in Example E above) and propionamide oxime (prepared accordingto the procedures described in J. Org. Chem., 46, 3953 (1981)), thetitle compound was prepared as a semisolid. The reaction was monitoredby tlc on silica gel (Rf=0.2 in 50% ethyl acetate/hexane) andpurification was by preparative plate chromatography (silica gel using50% ethyl acetate/hexane as the eluant).

NMR data was as follows:

¹H-nmr (d⁶-DMSO): δ=7.27 (d, 1H), 6.83 (s, 1H), 6.64 (d, 1H), 6.47 (d,1H), 6.38 (broad s, 2H), 4.24 (m, 1H), 2.07 (q, 2H), 1.41 (d, 3H).

C₁₂H₁₅Cl₂N₃O₂ (MW=304.17); mass spectroscopy (MH⁺) 305.

Example 50 Synthesis of N-(4-ethoxycarbonylphenyl)alanine iso-butylester (S isomer)

Following General Procedure M and using ethyl 4-aminobenzoate (Aldrich)and iso-butyl R-(+)-lactate (Aldrich), the title compound was preparedas an oil. The reaction was monitored by silica gel tlc (Rf=0.21 in 10%EtOAc/hexanes). Purification was by preparative plate thin layerchromatography using 25% EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.82 (d, J=8.73 Hz, 2H), 6.51 (d, J=8.79 Hz, 2H), 4.81(d, J=7.82 Hz, 1H), 4.25 (q, J=7.14 Hz, 2H), 4.15 (quint, J=7.40 Hz,1H), 3.87 (m, 2H), 1.87 (sept, J=6.70 Hz, 1H), 1.43 (d, J=6.95 Hz, 3H),1.30 (t, J=7.14 Hz, 3H), 0.84 (d, J=6.71 Hz, 6H).

¹³C-nmr (CDCl₃): δ=174.5, 167.3, 151.0, 132.0, 119.9, 112.5, 71.9, 60.8,51.9, 28.2, 19.5, 19.2, 15.0.

C₁₆H₂₃NO₄ (MW=293.37); mass spectroscopy (MH⁺) 294.

Example 51 Synthesis of N-[3,5-di(trifluoromethyI)phenyl]alanineiso-butyl ester (S isomer)

Following General Procedure M and using 3,5-di(trifluoromethyl)aniline(Aldrich) and iso-butyl R-(+)-lactate (Aldrich), the title compound wasprepared as an oil. The reaction was monitored by silica gel tlc(Rf=0.38 in 10% EtOAc/hexanes). Purification was by preparative platethin layer chromatography using 10% EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=7.13 (s, 1H), 6.91 (s, 2H), 4.97 (d, J=8.24 Hz, 1H),4.18 (m, 1H), 3.93 (d, J=6.59 Hz, 2H), 1.93 (sept, J=6.71 Hz, 1H), 1.49(d, J=7.02 Hz, 3H), 0.89 (d, J=6.59 Hz, 6H).

¹³C-nmr (CDCl₃): δ=174.4, 147.9, 133.6, 133.2, 132.7, 132.3, 129.4,125.8, 122.2, 118.6, 112.81, 112.76, 111.42, 111.37, 111.32, 111.27,111.22, 72.2, 52.0, 32.1, 28.24, 28.17, 23.2, 19.5, 19.3, 19.2, 18.9,14.6.

C₁₅H₁₇F₆NO₂ (MW=357.30); mass spectroscopy (MH⁺) 358.

Example 52 Synthesis of N-(3,5-dimethoxyphenyl)alanine iso-butyl ester

N-(3,5-dimethoxyphenyl)alanine (crude, 454 mg) (prepared according tothe procedure described in U.S. Pat. No. 3,598,859 using3,5-dimethoxyaniline (Aldrich) and 2-chloropropionic acid (Aldrich)) wastreated in dry iso-butanol (10 mL) with 0.1 mL of chlorotrimethylsilaneand the reaction mixture refluxed overnight. The excess alcohol wasremoved at reduced pressure and the residue dissolved in ethyl acetate.The ethyl acetate solution was washed with saturated aqueous NaHCO₃,dried with Na₂SO₄ and the solvent removed to provide the title compound.The reaction was monitored by silica gel tlc (Rf=0.3 in 20%EtOAc/hexanes). Purification was by preparative thin layerchromatography using 20% EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (CDCl₃): δ=0.9 (d, J=7, 6H), 1.47 (d, J=7, 3H), 1.9-2.0 (m, 1H),3.7 (s, 6H), 3.85-4.0 (m, 2H), 4.1-4.2 (m, 1H), 4.3 (brs, 1H), 5.8 (s,2H), 5.9 (s, 1H).

¹³C-nmr (CDCl₃): δ=19.49, 19.52, 19.54, 28.3, 52.5, 55.6, 71.7, 91.1,92.7, 149.2, 162.3, 175.2.

C₁₅H₂₃NO₄ (MW=281.35).

Example 53 Synthesis of N-(2-napthyl)alanine O-acylpropionamidoximeester

Following General Procedure S and using N-(2-naphthyl)alanine2,4,5-trichlorophenyl ester (from Example E above) and propionamideoxime (prepared according to the procedures described in J. Org. Chem.,46, 3953 (1981)), the title compound was prepared. The reaction wasmonitored by silica gel tlc (Rf=0.5 in EtOAc). Purification was bysilica gel chromatography using 1:1 EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (DMSO-d₆): δ=1.03 (t, 3H), 1.45 (d, 3H).

C₁₆H₁₉N₃O₂ (MW=285.35); mass spectroscopy (M⁺) 285.

Example 54 Synthesis of N-(2-napthyl)alanine O-acylbutyramidoxime ester

Following General Procedure S and using N-(2-naphthyl)alanine2,4,5-trichlorophenyl ester (from Example E above) and butyramide oximeprepared according to the procedures described in J. Org. Chem., 46,3953 (1981)), the title compound was prepared as an oil. The reactionwas monitored by silica gel tlc (Rf=0.6 in EtOAc). Purification was bysilica gel chromatography using 1:1 EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (DMSO-d₆): δ=0.86 (t, 3H), 1.46 (d, 3H).

C₁₇H₂₁N₃O₂ (MW=299.37); mass spectroscopy (MH⁺) 299.

Example 55 Synthesis of N-(2-napthyl)alanine O-acylisovaleramidoximeester

Following General Procedure S and using N-(2-naphthyl)alanine2,4,5-trichlorophenyl ester (from Example E above) and isovaleramideoxime (prepared according to the procedures described in J. Org. Chem.,46, 3953 (1981)), the title compound was prepared as an oil. Thereaction was monitored by silica gel tlc (Rf=0.3 in 1:1 EtOAc/hexanes).Purification was by silica gel chromatography using 1:1 EtOAc/hexanes asthe eluant.

NMR data was as follows:

¹H-nmr (DMSO-d₆): δ=0.86 (t, 3H), 1.45 (d, 3H).

C₁₈H₂₃N₃O₂ (MW=313.40); mass spectroscopy (MH⁺) 313.

Example 56 Synthesis of N-(2-napthyl)alanine O-acylbenzamidoxime ester

Following General Procedure S and using N-(2-naphthyl)alanine2,4,5-trichlorophenyl ester (from Example E above) and benzamide oxime(prepared according to the procedures described in J. Org. Chem., 46,3953 (1981)), the title compound was prepared as an oil. The reactionwas monitored by silica gel tlc (Rf=0.3 in 1:1 EtOAc/hexanes).Purification was by silica gel chromatography using 1:1 EtOAc/hexanes asthe eluant.

NMR data was as follows:

¹H-nmr (DMSO-d₆): δ=4.42 (m, 1H), 1.53 (d, 3H).

C₂₀H₁₉N₃O₂ (MW=333.39); mass spectroscopy (MH⁺) 333.

Example 57 Synthesis of N-(2-napthyl)alanineO-acylcyclopropanecarboxamidoxime ester

Following General Procedure S and using N-(2-naphthyl)alanine2,4,5-trichlorophenyl ester (from Example E above) andcyclopropanecarboxamide oxime (prepared according to the proceduresdescribed in J. Org. Chem., 46, 3953 (1981)), the title compound wasprepared as an oil. The reaction was monitored by silica gel tlc (Rf=0.3in 1:1 EtOAc/hexanes). Purification was by silica gel chromatographyusing 1:1 EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (DMSO-d₆): δ=0.85 (m, 4H), 1.43 (d, 3H).

C₁₇H₁₉N₃O₂ (MW=297.36); mass spectroscopy (MH⁺) 297.

Example 58 Synthesis of N-(2-napthyl)alanineO-acylcyclopropylacetamidoxime ester

Following General Procedure S and using N-(2-naphthyl)alanine2,4,5-trichlorophenyl ester (from Example E above) andcyclopropylacetamide oxime (prepared according to the proceduresdescribed in J. Org. Chem., 46, 3953 (1981)), the title compound wasprepared as an oil. The reaction was monitored by silica gel tlc (Rf=0.3in 1:1 EtOAc/hexanes). Purification was by silica gel chromatographyusing 1:1 EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (DMSO-d₆): δ=1.43 (d, 3H), 1.91 (d, 2H).

C₁₈H₂₁N₃O₂ (MW=311.39); mass spectroscopy (MH⁺) 311.

Example 59 Synthesis of N-(2-napthyl)alanineO-acylcyclopentanecarboxamidoxime ester

Following General Procedure S and using N-(2-naphthyl)alanine2,4,5-trichlorophenyl ester (from Example E above) andcyclopentanecarboxamide oxime (prepared according to the proceduresdescribed in J. Org. Chem., 46, 3953 (1981)), the title compound wasprepared as an oil. The reaction was monitored by silica gel tlc (Rf=0.3in 1:1 EtOAc/hexanes). Purification was by silica gel chromatographyusing 1:1 EtOAc/hexanes as the eluant.

NMR data was as follows:

¹H-nmr (DMSO-d₆): δ=1.43 (d, 3H), 2.43 (m, 1H).

C₁₇H₁₉N₃O₂ (MW=297.36).

Example 60 Cellular Screen for the Detection of Inhibitors of β-AmyloidProduction

Numerous compounds of formula I above were assayed for their ability toinhibit β-amyloid production in a cell line possessing the Swedishmutation. This screening assay employed cells (K293=human kidney cellline) which were stably transfected with the gene for amyloid precursorprotein 751 (APP751) containing the double mutation Lys₆₅₁Met₆₅₂ toAsn₆₅₁Leu₆₅₂ (APP751 numbering) in the manner described in InternationalPatent Application Publication No. 94/10569⁸ and Citron et al.¹². Thismutation is commonly called the Swedish mutation and the cells,designated as “293 751 SWE”, were plated in Corning 96-well plates at1.5-2.5×10⁴ cells per well in Dulbecco's minimal essential media (Sigma,St. Louis, Mo.) plus 10% fetal bovine serum. Cell number is important inorder to achieve β-amyloid ELISA results within the linear range of theassay (˜0.2 to 2.5 ng per mL).

Following overnight incubation at 37° C. in an incubator equilibratedwith 10% carbon dioxide, media were removed and replaced with 200 μL ofa compound of formula I (drug) containing media per well for a two hourpretreatment period and cells were incubated as above. Drug stocks wereprepared in 100% dimethyl sulfoxide such that at the final drugconcentration used in the treatment, the concentration of dimethylsulfoxide did not exceed 0.5% and, in fact, usually equaled 0.1%.

At the end of the pretreatment period, the media were again removed andreplaced with fresh drug containing media as above and cells wereincubated for an additional two hours. After treatment, plates werecentrifuged in a Beckman GPR at 1200 rpm for five minutes at roomtemperature to pellet cellular debris from the conditioned media. Fromeach well, 100 μL of conditioned media or appropriate dilutions thereofwere transferred into an ELISA plate precoated with antibody 266 [P.Seubert, Nature (1992) 359:325-327] against amino acids 13-28 ofβ-amyloid peptide as described in International Patent ApplicationPublication No. 94/10569⁸ and stored at 4° C. overnight. An ELISA assayemploying labelled antibody 6C6 [P. Seubert, Nature (1992) 359:325-327]against amino acids 1-16 of β-amyloid peptide was run the next day tomeasure the amount of β-amyloid peptide produced.

Cytotoxic effects of the compounds were measured by a modification ofthe method of Hansen, et al.¹³. To the cells remaining in the tissueculture plate was added 25 μL of a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrarolium bromide (MTT)(Sigma, St. Louis, Mo.) stock solution (5 mg/mL) to a finalconcentration of 1 mg/mL. Cells were incubated at 37° C. for one hour,and cellular activity was stopped by the addition of an equal volume ofMTT lysis buffer (20% w/v sodium dodecylsulfate in 50%dimethylformamide, pH 4.7). Complete extraction was achieved byovernight shaking at room temperature. The difference in the OD_(562 nm)and the OD_(650 nm) was measured in a Molecular Device's UV_(max)microplate reader as an indicator of the cellular viability.

The results of the β-amyloid peptide ELISA were fit to a standard curveand expressed as ng/mL β-amyloid peptide. In order to normalize forcytotoxicity, these results were divided by the MTT results andexpressed as a percentage of the results from a drug free control. Allresults are the mean and standard deviation of at least six replicateassays.

The test compounds were assayed for β-amyloid peptide productioninhibition activity in cells using this assay. The results of this assaydemonstrate that, each of the compounds of Examples 1-59 inhibit theβ-amyloid peptide production by at least 30% as compared to control.

Example 61 In Vivo Suppression of β-Amyloid Release and/or Synthesis

This example illustrates how the compounds of this invention could betested for in vivo suppression of β-amyloid release and/or synthesis.For these experiments, 3 to 4 month old PDAPP mice are used [Games etal., (1995) Nature 373:523-527]. Depending upon which compound is beingtested, the compound is usually formulated at either 5 or 10 mg/ml.Because of the low solubility factors of the compounds, they may beformulated with various vehicles, such as corn oil (Safeway, South SanFrancisco, Calif.); 10% ethanol in corn oil;2-hydroxypropyl-β-cyclodextrin (Research Biochemicals International,Natick Mass.); and carboxy-methyl-cellulose (Sigma Chemical Co., St.Louis Mo.).

The mice are dosed subcutaneously with a 26 gauge needle and 3 hourslater the animals are euthanized via CO₂ narcosis and blood is taken bycardiac puncture using a 1 cc 25 G ⅝″ tuberculin syringe/needle coatedwith solution of 0.5 M EDTA, pH 8.0. The blood is placed in aBecton-Dickinson vacutainer tube containing EDTA and spun down for 15minutes at 1500×g at 5° C. The brains of the mice are then removed andthe cortex and hippocampus are dissected out and placed on ice.

1. Brain Assay

To prepare hippocampal and cortical tissue for enzyme-linkedimmunosorbent assays (ELISAs) each brain region is homogenized in 10volumes of ice cold guanidine buffer (5.0 M guanidine-HCl, 50 mMTris-HCl, pH 8.0) using a Kontes motorized pestle (Fisher, PittsburghPa.). The homogenates are gently rocked on a rotating platform for threeto four hours at room temperature and stored at −20° C. prior toquantitation of β-amyloid.

The brain homogenates are diluted 1:10 with ice-cold casein buffer[0.25% casein, phosphate buffered saline (PBS), 0.05% sodium azide, 20μg/ml aprotinin, 5 mM EDTA, pH 8.0, 10 μg/ml leupeptin], therebyreducing the final concentration of guanidine to 0.5 M, beforecentrifugation at 16,000×g for 20 minutes at 4° C. The β-amyloidstandards (1-40 or 1-42 amino acids) were prepared such that the finalcomposition equaled 0.5 M guanidine in the presence of 0.1% bovine serumalbumin (BSA).

The total β-amyloid sandwich ELISA, quantitating both β-amyloid (aa1-40) and β-amyloid (aa 1-42) consists of two monoclonal antibodies(mAb) to β-amyloid. The capture antibody, 266 [P. Seubert, Nature (1992)359:325-327], is specific to amino acids 13-28 of β-amyloid. Theantibody 3D6 [Johnson-Wood et al., PNAS USA (1997) 94:1550-1555], whichis specific to amino acids 1-5 of β-amyloid, is biotinylated and servedas the reporter antibody in the assay. The 3D6 biotinylation procedureemploys the manufacturer's (Pierce, Rockford Ill.) protocol forNHS-biotin labeling of immunoglobulins except that 100 mM sodiumbicarbonate, pH 8.5 buffer is used. The 3D6 antibody does not recognizesecreted amyloid precursor protein (APP) or full-length APP but detectsonly β-amyloid species with an amino terminal aspartic acid. The assayhas a lower limit of sensitivity of ˜50 pg/ml (11 pM) and shows nocross-reactivity to the endogenous murine β-amyloid peptide atconcentrations up to 1 ng/ml.

The configuration of the sandwich ELISA quantitating the level ofβ-amyloid (aa 1-42) employs the mAb 21F12 [Johnson-Wood et al., PNAS USA(1997) 94:1550-1555] (which recognizes amino acids 33-42 of β-amyloid)as the capture antibody. Biotinylated 3D6 is also the reporter antibodyin this assay which has a lower limit of sensitivity of ˜125 pg/ml (28pM).

The 266 and 21F12 capture mAbs are coated at 10 μg/ml into 96 wellimmunoassay plates (Costar, Cambidge Mass.) overnight at roomtemperature. The plates are then aspirated and blocked with 0.25% humanserum albumin in PBS buffer for at least 1 hour at room temperature,then stored desiccated at 4° C. until use. The plates are rehydratedwith wash buffer (Tris-buffered saline, 0.05% Tween 20) prior to use.The samples and standards are added to the plates and incubatedovernight at 4° C. The plates are washed ≧3 times with wash bufferbetween each step of the assay. The biotinylated 3D6, diluted to 0.5μg/ml in casein incubation buffer (0.25% casein, PBS, 0.05% Tween 20, pH7.4) is incubated in the well for 1 hour at room temperature. Avidin-HRP(Vector, Burlingame Calif.) diluted 1:4000 in casein incubation bufferis added to the wells for 1 hour at room temperature. The colorimetricsubstrate, Slow TMB-ELISA (Pierce, Cambridge Mass.), is added andallowed to react for 15 minutes, after which the enzymatic reaction isstopped with addition of 2 N H₂SO₄. Reaction product is quantified usinga Molecular Devices Vmax (Molecular Devices, Menlo Park Calif.)measuring the difference in absorbance at 450 nm and 650 nm.

2. Blood Assay

The EDTA plasma is diluted 1:1 in specimen diluent (0.2 gm/l sodiumphosphate.H₂O (monobasic), 2.16 gm/l sodium phosphate.7H₂O (dibasic),0.5 gm/l thimerosal, 8.5 gm/l sodium chloride, 0.5 ml Triton X-405, 6.0g/l globulin-free bovine serum albumin; and water). The samples andstandards in specimen diluent are assayed using the total β-amyloidassay (266 capture/3D6 reporter) described above for the brain assayexcept the specimen diluent was used instead of the casein diluentsdescribed.

From the foregoing description, various modifications and changes in thecomposition and method will occur to those skilled in the art. All suchmodifications coming within the scope of the appended claims areintended to be included therein.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 1(2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 43 amino acids (B) TYPE: peptide (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO 1: Asp AlaGlu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 LeuVal Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 GlyLeu Met Val Gly Gly Val Val Ile Ala Thr 35 40

What is claimed is:
 1. A pharmaceutical composition comprising apharmaceutically inert carrier and a pharmaceutically effective amountof a compound of formula I:

wherein R¹ is selected from the group consisting of: (a) a substitutedphenyl group of formula II:

 wherein R^(c) is selected from the group consisting of acyl, alkyl,alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen,nitro, trihalomethyl, thioalkoxy, and where R^(b) and R^(c) are fused toform a heteroaryl or heterocyclic ring with the phenyl ring wherein theheteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from1 to 3 are heteroatoms independently selected from the group consistingof oxygen, nitrogen and sulfur; R^(b) and R^(b′) are independentlyselected from the group consisting of hydrogen, halo, nitro, cyano,trihalomethyl, alkoxy, and thioalkoxy with the proviso that R^(b),R^(b′) and R^(c) are not all hydrogen and with the further proviso thatwhen R^(c) is hydrogen, then neither R^(b) nor R^(b′) are hydrogen; (b)2-naphthyl; and (c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8positions with 1 to 5 substituents selected from the group consisting ofalkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy; R² isselected from the group consisting of hydrogen, alkyl of from 1 to 4carbon atoms, alkylalkoxy of from 1 to 4 carbon atoms andalkylthioalkoxy of from 1 to 4 carbon atoms; and R³ is selected from thegroup consisting of: (a) —Y(CH₂)_(n)CHR⁴R⁵ wherein n is an integer offrom 0 to 2, Y is selected from the group consisting of oxygen andsulfur, R⁴ and R⁵ are independently selected from the group consistingof hydrogen, alkyl, alkenyl, aryl optionally substituted with from 1 to3 substituents selected from the group consisting of alkyl, alkoxy,halo, cyano, nitro, trihalomethyl, and thioalkoxy, heteroaryl optionallysubstituted with from 1 to 3 substituents selected from the groupconsisting of alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, andthioalkoxy, and where R⁴ and R⁵ are joined to form a cycloalkyl group, acycloalkenyl group or a heterocyclic group; (b) —ON═C(NH₂)R⁶ where R⁶ isselected from the group consisting of alkyl, aryl, cycloalkyl, andheteroaryl; (c) —O(CH₂)_(p)C(O)OR⁷ wherein p is an integer of from 1 to2 and R⁷ is alkyl; and (d) —NR⁸R⁹ wherein R⁸ and R⁹ are joined to form apyrrolyl group; and pharmaceutically acceptable salts thereof with theprovisos that
 1. when R¹ is the substituted phenyl group of formula IIabove, R^(b′) is hydrogen, R^(b) and R^(c) are chloro, and R² is methyl,then R³ is not —OCH(CH₃)—φ;
 2. when R¹ is the substituted phenyl groupof formula II above, when R^(b′) is hydrogen, R^(b) and R^(c) arechloro, and R³ is —OCH₂CH₃ then R² is not hydrogen;
 3. when R¹ is thesubstituted phenyl group of formula II above, R^(b′) is hydrogen, R^(b)and R^(c) are chloro, and R³ is —OCH₂CH(CH₃)₂ then R² is not—CH(CH₃)CH₂CH₃; and
 4. when R¹ is N-methylindol-5-yl and R² is methyl,then R³ is not —OCH₂CH₃.
 2. The pharmaceutical composition according toclaim 1 wherein R¹ is selected from the group consisting of4-substituted, 3,5-disubstituted or 3,4-disubstituted phenylsubstituents wherein the substituents at the 3 and/or 5 positions aredefined by R^(b), R^(b′) as above and the substituent at the 4 positionis defined by R^(c) as above.
 3. The pharmaceutical compositionaccording to claim 2 wherein R¹ is a 3,5-disubstituted phenyl selectedfrom the group consisting of 3,5-dichlorophenyl, 3,5-difluorophenyl,3,5-di(trifluoromethyl)phenyl and 3,5-dimethoxyphenyl.
 4. Thepharmaceutical composition according to claim 2 wherein R¹ is a3,4-disubstituted phenyl selected from the group consisting of3,4-dichlorophenyl, 3,4-difluorophenyl,3-(trifluoromethyl)-4-chlorophenyl, 3-chloro-4-cyanophenyl,3-chloro-4-iodophenyl, 3,4-ethylenedioxyphenyl, and3,4-methylenedioxyphenyl.
 5. The pharmaceutical composition according toclaim 2 wherein R¹ is a 4-substituted phenyl selected from the groupconsisting of 4-azidophenyl, 4-bromophenyl, 4-chlorophenyl,4-cyanophenyl, 4-ethylphenyl, 4-fluorophenyl, 4-iodophenyl,4-(phenylcarbonyl)phenyl, 4-(1-ethoxy)ethylphenyl, and4-(ethoxycarbonyl)phenyl.
 6. The pharmaceutical composition according toclaim 1 wherein R¹ is selected from the group consisting of 2-naphthyl,2-methylquinolin-6-yl, benzothiazol-6-yl and 5-indolyl.
 7. Thepharmaceutical composition according to claim 1 wherein R² is selectedfrom the group consisting of alkyl of from 1 to 4 carbon atoms,alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to4 carbon atoms.
 8. The pharmaceutical composition according to claim 7wherein R² is selected from the group consisting of methyl, ethyl,n-propyl and iso-butyl.
 9. The pharmaceutical composition according toclaim 1 wherein R³ is selected from the group consisting of methoxy,ethoxy, iso-propoxy, n-propoxy, n-butoxy, iso-butoxy, cyclopentoxy,allyloxy, 4-methylpentoxy, —O—CH₂—(2,2-dimethyl- 1,3-dioxolan-4-yl),—O—CH₂-cyclohexyl, —O—CH₂-(3-tetrahydrofuranyl),—O—CH₂—C(O)O-tert-butyl, —O—CH₂—C(CH₃)₃, —O—CH₂—φ, —OCH₂CH(CH₂CH₃)₂,—O(CH₂)₃CH(CH₃)₂, —ON═C(NH₂)φ, —ON═C(NH₂)CH₃, —ON═C(NH₂)CH₂CH₃,—ON═C(NH₂)CH₂CH₂CH₃, —ON═C(NH₂)-cyclopropyl, —ON═C(NH₂)—CH₂-cyclopropyl,—ON═C(NH₂)-cyclopentyl, and —ON═C(NH₂)CH₂CH(CH₃)₂.
 10. Thepharmaceutical composition according to claim 1 wherein the compound offormula I is selected from the group consisting of:N-(3,4-dichlorophenyl)alanine ethyl ester;N-(3-trifluoromethyl-4-chlorophenyl)alanine ethyl ester;N-(3,5-dichlorophenyl)alanine ethyl ester; N-(3,4-difluorophenyl)alanineethyl ester; N-(3,4-dichlorophenyl)alanine benzyl ester;N-(3,4-dichlorophenyl)alanine iso-butyl ester;N-(3,4-dichlorophenyl)alanine iso-propyl ester;N-(3,4-dichlorophenyl)alanine n-butyl ester;N-(3,4-dichlorophenyl)alanine methyl ester;N-(3,4-dichlorophenyl)alanine cyclopentyl ester;N-(3,4-dichlorophenyl)alanine n-propyl ester;N-(3,4-dichlorophenyl)alanine allyl ester; N-(3,4-dichlorophenyl)alanine4-methylpentyl ester; N-(3,4-dichlorophenyl)alanine2,2-dimethyl-1,3-dioxolane-4-methyl ester; N-(3,4-dichlorophenyl)alaninecyclohexylmethyl ester; N-(3,4-dichlorophenyl)alaninetert-butoxycarbonylmethyl ester; N-(3,4-dichlorophenyl)leucine iso-butylester; 2-[N-(3,4-dichlorophenyl)amino]pentanoic acid iso-butyl ester;N-(4-cyanophenyl)alanine iso-butyl ester;N-(3-chloro-4-cyanophenyl)alanine iso-butyl ester;N-(3,4-dichlorophenyl)alanine tetrahydrofuran-3-yl-methyl ester;N-(3-chloro-4-iodophenyl)alanine iso-butyl ester;2-[N-(3,4-dichlorophenyl)amino]butanoic acid iso-butyl ester;N-(4-chlorophenyl)alanine iso-butyl ester; N-(3,5-dichlorophenyl)alanineiso-butyl ester; N-(4-ethylphenyl)alanine methyl ester;N-[4-(1-ethoxy)ethylphenyl]alanine methyl ester;N-(3,4-dichlorophenyl)alanine 2,2-dimethylpropyl ester;N-(3,4-dichlorophenyl)glycine iso-butyl ester;N-(3,4-dichlorophenyl)alanine 2-ethylbuty ester;N-(3-chloro-4-iodophenyl)alanine iso-butyl ester;N-(4-azidophenyl)alanine iso-butyl ester;N-[(4-phenylcarbonyl)phenyl]alanine iso-butyl ester;N-(3,5-difluorophenyl)alanine iso-butyl ester;N-(3,4-dichlorophenyl)alanine O-acylacetamidoxime ester;N-(3,4-dichlorophenyl)alanine pyrrolyl amide;N-(3,4-dichlorophenyl)alanine O-acylpropionamideoxime ester;N-(3,4-dichlorophenyl)alanine O-acylbutyramideoxime ester;2-[N-(naphth-2-yl)amino]butanoic acid ethyl ester;N-(naphth-2-yl)alanine iso-butyl ester; N-(2-methylquinolin-6-yl)alanine iso-butyl ester; N-(3,4-ethylenedioxyphenyl)alanine iso-butylester; N-(3,4-methylenedioxyphenyl)alanine iso-butyl ester;N-(naphth-2-yl)alanine methyl ester; N-(benzothiazol-6-yl)alanine ethylester; N-(indol-5-yl)alanine iso-butyl ester; N-(naphth-2-yl)alanineO-acylacetamidoxime ester; N-(2-naphthyl)alanine ethyl ester;N-(4-ethoxycarbonylphenyl)alanine iso-butyl ester;N-(3,5-di(trifluoromethyl)phenyl)alanine iso-butyl ester;N-(3,5-dimethoxyphenyl)alanine iso-butyl ester; N-(2-napthyl)alanineO-acylpropionamidoxime ester; N-(2-napthyl)alanine O-acylbutyramidoximeester; N-(2-napthyl)alanine O-acylisovaleramidoxime ester;N-(2-napthyl)alanine O-acylbenzamidoxime ester; N-(2-napthyl)alanineO-acylcyclopropanecarboxamidoxime ester; N-(2-napthyl)alanineO-acylcyclopropylacetamidoxime ester; and N-(2-napthyl)alanineO-acylcyclopentanecarboxamidoxime ester.
 11. A compound of formula III:

wherein R¹ is selected from the group consisting of: (a) a substitutedphenyl group of formula II:

 wherein R^(c) is selected from the group consisting of acyl, alkyl,alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen,nitro, trihalomethyl, thioalkoxy, and where R^(b) and R^(c) are fused toform a heteroaryl or heterocyclic ring with the phenyl ring wherein theheteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from1 to 3 are heteroatoms independently selected from the group consistingof oxygen, nitrogen and sulfur; R^(b) and R^(b′) are independentlyselected from the group consisting of hydrogen, halo, nitro, cyano,trihalomethyl, alkoxy, and thioalkoxy with the proviso that R^(b),R^(b′) and R^(c) are not all hydrogen and with the further proviso thatwhen R^(c) is hydrogen, then neither R^(b) nor R^(b′) are hydrogen; (b)2-naphthyl; and (c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8positions with 1 to 5 substituents selected from the group consisting ofalkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy; R² isselected from the group consisting of alkyl of from 1 to 4 carbon atoms,alkylalkoxy of from 1 to 4 carbon atoms and alkylthioalkoxy of from 1 to4 carbon atoms; and R³ is selected from the group consisting of: (a)—Y(CH₂)_(n)CHR⁴R⁵ wherein n is an integer of from 0 to 2, Y is selectedfrom the group consisting of oxygen and sulfur, R⁴ and R⁵ areindependently selected from the group consisting of hydrogen, alkyl,alkenyl, aryl optionally substituted with from 1 to 3 substituentsselected from the group consisting of alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy, heteroaryl optionally substituted withfrom 1 to 3 substituents selected from the group consisting of alkyl,alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy, and where R⁴and R⁵ are joined to form a cycloalkyl group, a cycloalkenyl group or aheterocyclic group; (b) —ON═C(NH₂)R⁶ where R⁶ is selected from the groupconsisting of alkyl, aryl, cycloalkyl, and heteroaryl; (c)—O(CH₂)_(p)C(O)OR⁷ wherein p is an integer of from 1 to 2 and R⁷ isalkyl; (d) —NR⁸R⁹ wherein R⁸ and R⁹ are joined to form a pyrrolyl group;and pharmaceutically acceptable salts thereof with the proviso excludingthe following compounds:
 1. when R¹ is the substituted phenyl group offormula II above, R^(b′) is hydrogen, R^(b) and R^(c) are chloro, and R²is methyl, then R³ is not —OCH(CH₃)—φ;
 2. when R¹ is the substitutedphenyl group of formula II above, when R^(b′) is hydrogen, R^(b) andR^(c) are chloro, and R³ is —OCH₂CH₃ then R² is not hydrogen;
 3. when R¹is the substituted phenyl group of formula II above, R^(b′) is hydrogen,R^(b) and R^(c) are chloro, and R³ is —OCH₂CH(CH₃)₂ then R² is not—CH(CH₃)CH₂CH₃;
 4. when R¹ is a substituted phenyl group of the formula:

wherein X is hydrogen, halogen or methyl and Y is halogen or methyl,then R² is not hydrogen or methyl;
 5. when R¹ is 2-napthyl or a6-methoxy-substituted 2-naphthyl and R² is methyl then R³ is not—OCH₂CH₃; and
 6. when R¹ is N-methylindol-5-yl and R² is methyl, then R³is not —OCH₂CH₃; and still with further proviso excluding the followingknown compounds: N-(4-chlorophenyl)alanine ethyl ester;N-(3,4-dichlorophenyl)alanine ethyl ester; N-(3,5-dichlorophenyl)alanineethyl ester; N-(4-n-butylphenyl)alanine ethyl ester;N-(3,4-dinitrophenyl)alanine ethyl ester; N-(4-chlorophenyl)glycineheptenyl ester; N-(4-methylphenyl)glycine butyl ester;N-(3-nitrophenyl)glycine decyl ester; N-(3,4-difluorophenyl)alaninemethyl ester; N-(3,4-difluorophenyl)alanine ethyl ester;N-(3,4-difluorophenyl)alanine iso-propyl ester;N-(4-fluorophenyl)alanine ethyl ester;N-(3-chloro-4-fluorophenyl)alanine methyl ester;N-(3-chloro-4-fluorophenyl)alanine ethyl ester; andN-(3-chloro-4-fluorophenyl)alanine iso-propyl ester.
 12. The compoundaccording to claim 11 wherein R¹ is selected from the group consistingof 4-substituted, 3,5-disubstituted or 3,4-disubstituted phenylsubstituents wherein the substituents at the 3 and/or 5 positions aredefined by R^(b), R^(b′) as above and the substituent at the 4 positionis defined by R^(c) as above.
 13. The compound according to claim 12wherein R¹ is a 3,5-disubstituted phenyl selected from the groupconsisting of 3,5-dichlorophenyl, 3,5-difluorophenyl,3,5-di(trifluoromethyl)phenyl and 3,5-dimethoxyphenyl.
 14. The compoundaccording to claim 11 wherein R¹ is a 3,4-disubstituted phenyl selectedfrom the group consisting of 3,4-dichlorophenyl, 3,4-difluorophenyl,3-(trifluoromethyl)-4-chlorophenyl, 3-chloro-4-cyanophenyl,3-chloro-4-iodophenyl, 3,4-ethylenedioxyphenyl, and3,4-methylenedioxyphenyl.
 15. The compound according to claim 11 whereinR¹ is a 4-substituted phenyl selected from the group consisting of4-azidophenyl, 4-bromophenyl, 4-chlorophenyl, 4-cyanophenyl,4-ethylphenyl, 4-fluorophenyl, 4-iodophenyl, 4-(phenylcarbonyl)phenyl,4-(1-ethoxy)ethylphenyl, and 4-(ethoxy-carbonyl)phenyl.
 16. The compoundaccording to claim 11 wherein R¹ is selected from the group consistingof 2-naphthyl, 2-methylquinolin-6-yl, benzothiazol-6-yl and 5-indolyl.17. The compound according to claim 11 wherein R² is selected from thegroup consisting of methyl, ethyl, n-propyl and iso-butyl.
 18. Thecompound according to claim 11 wherein R³ is selected from the groupconsisting of methoxy, ethoxy, iso-propoxy, n-propoxy, n-butoxy,iso-butoxy, cyclopentoxy, allyloxy, 4-methylpentoxy,—O—CH₂—(2,2-dimethyl-1,3-dioxolan-4-yl), —O—CH₂-cyclohexyl,—O—CH₂—(3-tetrahydrofuranyl), —O—CH₂—C(O)O-tert-butyl, —O—CH₂—C(CH₃)₃,and —OCH₂CH(CH₂CH₃)₂.
 19. The compound according to claim 11 wherein thecompound of formula I is selected from the group consisting of:N-(3-trifluoromethyl-4-chlorophenyl)alanine ethyl ester;N-(3,4-dichlorophenyl)alanine benzyl ester;N-(3,4-dichlorophenyl)alanine cyclopentyl ester;N-(3,4-dichlorophenyl)alanine allyl ester; N-(3,4-dichlorophenyl)alanine2,2-dimethyl-1,3-dioxolane-4-methyl ester; N-(3,4-dichlorophenyl)alaninecyclohexylmethyl ester; N-(3,4-dichlorophenyl)alaninetert-butoxycarbonylmethyl ester;2-{N-(3,4-dichlorophenyl)amino}pentanoic acid iso-butyl ester;N-(4-cyanophenyl)alanine iso-butyl ester;N-(3-chloro-4-cyanophenyl)alanine iso-butyl ester;N-(3,4-dichlorophenyl)alanine tetrahydrofuran-3-yl-methyl ester;2-{N-(3,4-dichlorophenyl)amino}butanoic acid iso-butyl ester;N-(4-ethylphenyl)alanine methyl ester;N-{4-(1-ethoxy)ethylphenyl}alanine methyl ester;N-(4-azidophenyl)alanine iso-butyl ester;N-{(4-phenylcarbonyl)phenyl}alanine iso-butyl ester;N-(3,4-dichlorophenyl)alanine O-acylacetamidoxime ester;N-(3,4-dichlorophenyl)alanine pyrrolyl amide;N-(3,4-dichlorophenyl)alanine O-acylpropionamideoxime ester;N-(3,4-dichlorophenyl)alanine O-acylbutyramideoxime ester;2-{N-(naphth-2-yl)amino}butanoic acid ethyl ester;N-(naphth-2-yl)alanine iso-butyl ester; N-(2-methylquinolin-6-yl)alanineiso-butyl ester; N-(3,4-ethylenedioxyphenyl)alanine iso-butyl ester;N-(3,4-methylenedioxyphenyl)alanine iso-butyl ester;N-(naphth-2-yl)alanine methyl ester; N-(benzothiazol-6-yl)alanine ethylester; N-(indol-5-yl)alanine iso-butyl ester; N-(naphth-2-yl)alanineO-acylacetamidoxime ester; N-(4-ethoxycarbonylphenyl)alanine iso-butylester; N-(3,5-di(trifluoromethyl)phenyl)alanine iso-butyl ester;N-(3,5-dimethoxyphenyl)alanine iso-butyl ester; N-(2-napthyl)alanineO-acylpropionamidoxime ester; N-(2-napthyl)alanine O-acylbutyramidoximeester; N-(2-napthyl)alanine O-acylisovaleramidoxime ester;N-(2-napthyl)alanine O-acylbenzamidoxime ester; N-(2-napthyl)alanineO-acylcyclopropanecarboxamidoxime ester; N-(2-napthyl)alanineO-acylcyclopropylacetamidoxime ester; and N-(2-napthyl)alanineO-acylcyclopentanecarboxamidoxime ester.
 20. A compound of formula III:

wherein R¹ is selected from the group consisting of: (a) a substitutedphenyl group of formula II:

 wherein R^(c) is selected from the group consisting of acyl, alkyl,alkoxy, alkoxycarbonyl, alkylalkoxy, azido, cyano, halo, hydrogen,nitro, trihalomethyl, thioalkoxy, and where R^(b) and R^(c) are fused toform a heteroaryl or heterocyclic ring with the phenyl ring wherein theheteroaryl or heterocyclic ring contains from 3 to 8 atoms of which from1 to 3 are heteroatoms independently selected from the group consistingof oxygen, nitrogen and sulfur; R^(b) and R^(b′) are independentlyselected from the group consisting of hydrogen, halo, nitro, cyano,trihalomethyl, alkoxy, and thioalkoxy with the proviso that R^(b),R^(b′) and R^(c) are not all hydrogen and with the further proviso thatwhen R^(c) is hydrogen, then neither R^(b) nor R^(b′) are hydrogen; (b)2-naphthyl; and (c) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8positions with 1 to 5 substituents selected from the group consisting ofalkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy; R² isalkyl selected from the group consisting of ethyl, n-propyl, iso-propyl,n-butyl, and iso-butyl; and R³ is selected from the group consisting of:(a) —Y(CH₂)_(n)CHR⁴R⁵ wherein n is an integer of from 0 to 2, Y isselected from the group consisting of oxygen and sulfur, R⁴ and R⁵ areindependently selected from the group consisting of hydrogen, alkyl,alkenyl, aryl optionally substituted with from 1 to 3 substituentsselected from the group consisting of alkyl, alkoxy, halo, cyano, nitro,trihalomethyl, and thioalkoxy, heteroaryl optionally substituted withfrom 1 to 3 substituents selected from the group consisting of alkyl,alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy, and where R⁴and R⁵ are joined to form a cycloalkyl group, a cycloalkenyl group or aheterocyclic group; (b) —ON═C(NH₂)R⁶ where R⁶ is selected from the groupconsisting of alkyl, aryl, cycloalkyl, and heteroaryl; (c)—O(CH₂)_(p)C(O)OR⁷ wherein p is an integer of from 1 to 2 and R⁷ isalkyl; (d) —NR⁸R⁹ wherein R⁸ and R⁹ are joined to form a pyrrolyl group;and pharmaceutically acceptable salts thereof.